Methods of microbial oil extraction and separation

ABSTRACT

Lipids can be extracted from a microbial biomass that constitutes at least 20% lipids by weight and has a moisture content of less than 4% by weight by applying pressure to the biomass so as to release lipids therefrom, thereby leaving a biomass of reduced lipid content; and collecting the lipids.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a national stage entry of International ApplicationNo. PCT/US10/31108, filed Apr. 14, 2010, which claims the benefit under35 U.S.C. 119(e) of U.S. Provisional Application No. 61/169,271, filedApr. 14, 2009, and U.S. Provisional Application No. 61/299,250, filedJan. 28, 2010. This application is also a continuation-in part ofInternational Application No. PCT/US2009/060692, filed Oct. 14, 2009which claims the benefit under 35 U.S.C. 119(e) of U.S. ProvisionalApplication No. 61/246,070, filed Sep. 25, 2009, U.S. ProvisionalApplication No. 61/173,166, filed Apr. 27, 2009, U.S. ProvisionalApplication No. 61/157,187, filed Mar. 3, 2009 and U.S. ProvisionalApplication No. 61/105,121, filed Oct. 14, 2008. This application isalso a continuation-in-part of International Application Nos.PCT/US2009/066142 and PCT/US2009/066141, both of which were filed Nov.30, 2009, and each of which claims the benefit under 35 U.S.C. 119(e) ofU.S. Provisional Application No. 61/219,525, filed Jun. 23, 2009, U.S.Provisional Application No. 61/174,357, filed Apr. 30, 2009, U.S.Provisional Application No. 61/118,994, filed Dec. 1, 2008, and U.S.Provisional Application No. 61/118,590, filed Nov. 28, 2008. Each ofthese applications is incorporated herein by reference in its entiretyfor all purposes.

REFERENCE TO A SEQUENCE LISTING

This application includes a sequence listing as shown in pages 1-21,appended hereto.

FIELD OF INVENTION

This invention generally relates to the production and extraction of oilfrom microorganisms. In particular, the invention provides methods forextracting, recovering, isolating, and obtaining oil from amicroorganism and compositions comprising the oil. The inventionaccordingly relates to the fields of biology, microbiology, fermentationtechnology and oil and fuel production technology.

BACKGROUND OF THE INVENTION

Fossil fuel is a general term for combustible geologic deposits oforganic materials formed from decayed plants and animals that have beenconverted to crude oil, coal, natural gas, or heavy oils by exposure toheat and pressure in the earth's crust over hundreds of millions ofyears.

Fossil fuels are a finite, non-renewable resource. With globalmodernization in the 20^(th) and 21^(st) centuries, the demand forenergy from fossil fuels, especially gasoline derived from oil, isgrowing and has been the cause of major regional and global conflicts.Increased demand for energy has also increased the cost of hydrocarbonfuels. Aside from energy, many industries, including the plastics andchemical manufacturing industries, are dependent on the availability ofhydrocarbons as a feedstock for manufacturing. Alternatives to currentsources of supply would help mitigate the upward pressure on these rawmaterial costs.

Lipids for use in biofuels can be produced in microorganisms, such asalgae, fungi, and bacteria. Typically, manufacturing a lipid in amicroorganism involves growing microorganisms, such as algae, fungi, orbacteria, which are capable of producing a desired lipid in a fermentoror bioreactor, isolating the microbial biomass, drying it, andextracting the intracellular lipids, which are a form of oil. However,these processes are generally considered to be inefficient andexpensive, particularly when one considers the scale on which they mustbe conducted to produce meaningful supplies of fuel. One significantproblem with these processes is the extraction of the lipid or oil froma microorganism.

There is a need for a process for extracting oil from microorganismsthat mitigates the problems of low efficiency and high cost of currentmethods for lipid extraction from microorganisms. The present inventionprovides such a process.

SUMMARY OF THE INVENTION

The present invention provides methods for extracting lipids/oil frommicrobial biomass. In one embodiment, the present invention provides amethod for extracting oil from microbial biomass, said method comprisingthe steps of subjecting dry microbial biomass having a moisture contentof less than 6% by weight and constituting at least 20% oil by weightand heat conditioned to a temperature in the range of 70° C. to 150° C.(160° F. to 300° F.) to pressure sufficient to extract more than 5% ofthe oil by weight from the biomass so that extracted oil and spentbiomass of reduced oil content is produced. In various embodiments, morethan 75% of the oil by weight in the dry microbial biomass is extractedfrom the biomass in the pressing step.

Thus, this method comprises drying and then conditioning the microbialbiomass to produce conditioned feedstock that is then subjected topressure. Conditioning changes the physical and/or physiochemicalproperties of the biomass but does not cause the release of more than 5%of the oil in the biomass. The conditioning step comprises heating thedry microbial biomass to a temperature in the range of 70° C. to 150° C.(160° F. to 300° F.), thereby altering its moisture content. In variousembodiments, a “bulking agent” or “press-aid” is added either to themicrobial biomass or to conditioned feedstock prior to the applicationof pressure during the pressing step. During the pressing step, theconditioned feedstock is subjected to pressure sufficient to separate atleast 5% of the oil in the biomass or conditioned feedstock from othercomponents. These other components are contained in the “spent biomass”,which may include residual oil but in any event has reduced oil contentrelative to the conditioned feedstock. In one embodiment, the pressureis exerted by an expeller press.

In various embodiments of this and other aspects of the invention, thebiomass is prepared by fermentation of a microbe selected from the groupconsisting of microalgae, oleaginous bacteria, oleaginous yeast, andfungi. In various embodiments, the microalgae is a species of a genusselected from Chlorella, Parachlorella, or Prototheca, or is one of theother species in Table 1, below. In various embodiments, the oleaginousbacteria is a species of the genus Rhodococcus. In various embodiments,the oleaginous yeast is Rhodotorula glutinis or another species listedin Table 2, below. In various embodiments, the fungi is a species listedin Table 3, below.

In various embodiments of this and other aspects of the invention, thebiomass is prepared by fermentation of a microbe that contains 18:1fatty acid. In various embodiments, the microbe has a fatty acid profileof less than 2% C14:0; about 13-16% C16:0; about 1-4% C18:0; about64-71% C18:1; about 10-15% C18:2; about 0.5-2% w/w C18:3; and less than1% carbon chain length 20 or longer. In various embodiments, the microbehas a fatty acid profile of about 1-2% C14:0; about 20% C16:0; about 4%C18:0; about 64% C18:1; and about 7-8% C18:2. In some embodiments, themicrobe has a fatty acid profile of about C14:0 (1.65); C16:0 (28.0);C18:0 (2.90); C18:1 (53.80); C18:2 (10.95); and C18:3alpha (0.80). Inother embodiments, the microbe has a fatty acid profile of C14:0 (2.33);C15:0 (9.08); C16:0 (24.56); C16:1 (11.07); C17:0 (10.50); C18:0 (2.49);C18:1 (17.41); C18:2 (0.05). In still other embodiments, the microbe hasa fatty acid profile of C12 (less than 1%); C14:0 (2.18-3.36); C15:0(0.12-0.25); C16:0 (29.94-33.26); C16:1 (0.49-0.76); C17:0; C18:0(6.88-8.17); C18:1 (42.68-48.12); C18:2 (7.88-9.28) C18:3 alpha(0.84-1.33); and greater than C:20 (1.1-1.45). In various embodiments,the microbe has less than 0.5% DHA. In these and other embodiments, themicrobe is, in some instances, a microalgae.

In various embodiments of this and other aspects of the invention, themicrobial biomass (dry or hydrated) or conditioned feedstock contains atleast 25% oil (lipids) by weight. In various embodiments, the drymicrobial biomass or conditioned feedstock contains at least 25% oil bydry cell weight. In various embodiments, the dry microbial biomass orconditioned feedstock contains at least 40%, at least 50%, or at least75% oil by dry cell weight. In various embodiments, the dry microbialbiomass or conditioned feedstock contains at least 15% carbohydrate bydry cell weight.

In various embodiments of this and other aspects of the invention, theconditioning step involves the application of heat and/or pressure tothe biomass. In various embodiments, the conditioning step comprisesheating the biomass at a temperature in the range of 70° C. to 150° C.(160° F. to 300° F.). In various embodiments, the heating is performedusing a vertical stacked shaker. In various embodiments, theconditioning step comprises treating the dry biomass with an expander orextruder to shape and/or homogenize the biomass. In various embodiments,the dry biomass or conditioned feedstock has a moisture content of lessthan 5% by weight. In various embodiments, the dry biomass orconditioned feedstock has a moisture content in the range of 0.1% and 5%by weight. In various embodiments, the dry biomass or conditionedfeedstock has a moisture content of less than 4% by weight. In variousembodiments, the dry biomass or conditioned feedstock has a moisturecontent in the range of 0.5% and 3.5% by weight. In various embodiments,the dry biomass or conditioned feedstock has a moisture content in therange of 0.1% and 3% by weight.

In various embodiments of this and other aspects of the invention, abulking agent is added to the microbial biomass, which may be either dryor hydrated (i.e., biomass that has not been dried or that containssignificant, i.e., more than 6% by weight, moisture, including biomassin fermentation broth that has not been subjected to any process toremove or separate water) microbial biomass or conditioned feedstockprior to the pressing step. In various embodiments, the bulking agenthas an average particle size of less than 1.5 mm. In variousembodiments, the bulking agent is selected from the group consisting ofcellulose, corn stover, dried rosemary, soybean hulls, spent biomass(biomass of reduced lipid content relative to the biomass from which itwas prepared), sugar cane bagasse, and switchgrass. In variousembodiments, the bulking agent is spent biomass that contains between50% and 80% polysaccharide by weight and/or less than 10% oil by weight.In various embodiments, the polysaccharide in the spent biomass used asa bulking agent contains 20-30 mole percent galactose, 55-65 molepercent glucose, and/or 5-15 mole percent mannose.

The invention provides various methods relating to the extraction of oilfrom microbial biomass that employ the bulking agents described above.In one method, hydrated microbial biomass suitable for oil extraction isprepared by adding a bulking agent to the biomass and drying the mixtureobtained thereby to a moisture content less than 6% by weight, therebyforming a dried bulking agent/biomass mixture. In another method, oil isextracted from microbial biomass by co-drying hydrated microbial biomasscontaining at least 20% oil by weight and a bulking agent to form adried bulking agent/biomass mixture; reducing the moisture content inthe mixture to less than 4% by weight; and pressing the reduced moisturecontent mixture to extract oil therefrom, thereby forming spent biomassof reduced lipid content. In another method, increased yields of oil areobtained from microbial biomass containing at least 20% lipid by weightby co-drying the microbial biomass with a bulking agent, because theco-dried mixture will, upon pressing, release more oil than can beobtained from the biomass under the same conditions in the absence of abulking agent. In various embodiments of these and other methods of theinvention, the hydrated microbial biomass is contained in fermentationbroth that has not been subjected to processes to separate or removewater from the biomass prior to adding the bulking agent to the biomass.Typically, the admixture of bulking agent and biomass is conditioned byheating to a temperature in the range of 70° C. to 150° C. (160° F. to300° F.) immediately prior to the pressing step.

In various embodiments of the different aspects of the invention, drymicrobial biomass, hydrated microbial biomass admixed with a bulkingagent, or conditioned feedstock, optionally comprising a bulking agent,is subjected to pressure in a pressing step to extract oil, producingoil separated from the spent biomass. The pressing step involvessubjecting pressure sufficient to extract oil from the conditionedfeedstock. Cell lysis will occur during this step, if the biomass orfeedstock has not been subjected to conditions that lyse some or all ofthe cells prior to the pressing step. In various embodiments of thedifferent aspects of the invention, the pressing step will involvesubjecting the conditioned feedstock to at least 10,000 psi of pressure.In various embodiments, the pressing step involves the application ofpressure for a first period of time, a reduction in pressure for asecond period of time, and then application of a pressure higher thanduring the first period of time for a third period of time. This processmay be repeated one or more times (“oscillating pressure”). In variousembodiments, more than 5 cycles of oscillating pressure are applied. Invarious embodiments, one or more of the subsequent cycles may exert anaverage pressure that is higher than the average pressure exerted in oneor more earlier cycles. For example and without limitation, the averagepressure in the last cycle can be at least 2-fold higher than theaverage pressure in the first or any earlier cycle. In variousembodiments, moisture content of the conditioned feedstock is controlledduring the pressing step. In various embodiments, the moisture iscontrolled in a range of from 0.1% to 3% by weight

In various embodiments, the pressing step is conducted with an expellerpress. In various embodiments, the pressing step is conducted in acontinuous flow mode. In various embodiments, the oiling rate is atleast 500 g/min to no more than 1000 g/min. In various continuous flowembodiments, the expeller press is a device comprising a continuouslyrotating worm shaft within a cage having a feeder at one end and a chokeat the opposite end, having openings within the cage is utilized. Theconditioned feedstock enters the cage through the feeder, and rotationof the worm shaft advances the feedstock along the cage and appliespressure to the feedstock disposed between the cage and the choke, thepressure releasing oil through the openings of cage and extruding spentbiomass from the choke end of the cage. In various embodiments, the cagehas an internal length that is between at least ten times to at leasttwenty times its internal diameter. In various embodiments, the cagecomprises a plurality of elongated bars with at least some of theelongated bars separated by one or more spacers, the bars resting on aframe, wherein the one or more spacers between the bars form theopenings, and oil is released through the openings to a collectingvessel fluidly coupled with the cage. In various embodiments, thespacers between the elongated bars are of different thicknesses therebyallowing variation of the space between each elongated bar. In variousembodiments, either the spacers or the gaps between the bars are from0.005 to 0.030 inches thick.

In various embodiments, the pressure increases by a factor of between 10and 20 from the feeder end to the choke end of the cage. In variousembodiments, the pressure along the cage does not increase by more than100% of the pressure at the feeder end of the cage per linear foot ofthe cage between the feeder and choke ends of the cage. In variousembodiments, the power consumed by the device does not increase by morethan 10% when fully loaded with conditioned feedstock relative torunning empty. In various embodiments, the residence time of feedstockin the barrel of the device is no longer than 5-10 min. In variousembodiments, either the temperature of the expeller device or thepressure exerted by the expeller device or both are monitored and/orcontrolled.

In various embodiments, pressure is controlled by adjusting rotationalvelocity of a worm shaft. In various embodiments, including those inwhich pressure is not controlled, an expeller (screw) press comprising aworm shaft and a barrel can be used. In various embodiments, the barrelhas a length and a channel having a diameter sized to receive the wormshaft, and wherein the barrel length is at least 10 to 15 times greaterthan the channel diameter. In various embodiments, the barrel of thepress has an entrance and an exit and the diameter of the worm shaftincreases from the entrance to the exit, and the pressing comprisesincreasing the pressure from the entrance to the exit of the barrel; invarious embodiments, the pressure at the exit is 12 to 16, or even up to20 times higher than the pressure at the entrance. In variousembodiments, the expeller press comprises a worm shaft and a barrelhaving a first channel and a second channel, both channels concentricand sized to receive the worm shaft, wherein the first channel has afirst diameter and the second channel has a second diameter differentthan the first diameter. In various embodiments, the conditionedfeedstock remains resident in the barrel of the expeller press for 5 to10 minutes.

In various embodiments, the expeller press comprises a worm shaftdisposed in a barrel lined with a plurality of elongate bars separatedby one or more spacers therebetween, the spacers creating a gap betweenthe elongate bars. In such a press, pressure can be controlled byadjusting the gap by changing the size or number of spacers between theelongate bars, and/or if the press has a space between an outer surfaceof the worm shaft and an inner surface of the elongate bars, pressurecan be controlled by replacing at least some of the elongate bars withdifferent sized bars so as to change the space. In various embodiments,the press comprises an output aperture and an adjustable choke coupledtherewith, and pressure is controlled by adjusting the choke to increaseor decrease the pressure. In various embodiments, the press comprises aworm shaft disposed in a barrel, and pressure is controlled by adjustinga gap between an outer surface of the worm shaft and an inside surfaceof the barrel.

After the pressing step, the method results in the extraction of oil andthe production of spent biomass. In various embodiments, the releasedoil contains solid particles of biomass or conditioned feedstock, andthe method further comprises separating the released oil from the solidparticles. Optionally, the separated solid particles can be subjected topressure to extract any remaining oil therefrom. In various embodiments,the extracted oil contains no more than 8 ppm chloride, no more than 2ppm phosphorus, no more than 26 ppm potassium, no more than 12 ppmsodium, and/or no more than 5 ppm sulfur. The oil produced by theprocess is useful in a variety of applications, including but notlimited to the production of fuels such as biodiesel and renewablediesel and the production of food.

In various embodiments, the oil content in the spent biomass of reducedoil content is at least 45 percent less than the oil content of themicrobial biomass before the pressing step. In various embodiments, thespent biomass of reduced oil content remaining after the pressing stepis pelletized or extruded as a cake. The spent biomass, which may besubjected to additional processes, including additional conditioning andpressing or solvent-based or other extraction methods to extractresidual oil, is similarly useful in a variety of applications,including but not limited to use as food, particularly for animals, andas a bulking agent. In various embodiments, remaining oil is extractedfrom the spent biomass of reduced oil content; in various embodimentsthe extracting is performed by subjecting the spent biomass to pressureor by extracting the oil with an organic solvent.

In view of the foregoing, the present invention is directed, in oneaspect, to a method for extracting lipids from microbial biomass. In oneembodiment, the method comprises subjecting microbial biomassconstituting at least 20% lipids by weight and having a moisture contentof less than 6% by weight to pressure, whereby cells of the biomass arelysed, releasing more than 5% of the lipids and leaving spent biomass ofreduced lipid content, wherein the extracted lipids and spent biomassare separated from each other.

In some cases, the microbial biomass is subjected to a lower pressurefor a first period of time followed by a higher pressure for a secondperiod of time. In some cases, the microbial biomass is subjected tomore than 5 cycles of oscillating pressure, and the average pressureexerted on the biomass during the course of the last cycle is at least 2fold higher than the average pressure exerted on the biomass during thecourse of the first cycle. In some cases, the microbial biomass issubject to pressure by a method comprising continuous flow through adevice applying the pressure. In one embodiment, the device is anexpeller press. In some cases, the microbial biomass is subjected to atleast 10,000 PSI of pressure.

In some embodiments, the microbial biomass is subject to pressure by amethod comprising continuous flow through a device applying thepressure, wherein the device is a continuously rotating worm shaftwithin a cage having a feeder at one end and a choke at an end oppositethereof, and having openings within the cage, wherein the biomass entersthe cage through the feeder, and rotation of the worm shaft advances thebiomass along the cage and applies pressure to the biomass disposedbetween the cage and the choke, the pressure lysing cells of the biomassand releasing oil through the openings of the cage such that spentbiomass of reduced oil content is extruded from the choke end of thecage. In some cases, the cage comprises a plurality of elongated barswith at least some of the elongated bars separated by one or morespacers, and the bars resting on a frame, wherein the one or morespacers between the bars form the openings, and lipids are releasedthrough the openings to a collecting vessel fluidly coupled with thecage. In some cases, the spacers between the elongated bars are ofdifferent thicknesses thereby allowing variation of the space betweeneach elongated bar. In some embodiments, either the spacers or the gapsbetween the bars are from 0.005 to 0.030 inches thick. In some cases,the pressure increases by a factor of between 10 and 20 from the feederend to the choke end of the cage. In some cases, the residence time ofbiomass in the barrel of the device is between 5-10 minutes. In someembodiments, the cage has an internal length that is between at leastten times to at least 20 times its internal diameter. In some cases, thepower consumed by a device does not increase by more than 10% when fullyloaded with microbial biomass relative to running empty. In some cases,the pressure along the cage does not increase by more than 100% of thepressure at the feeder end of the cage per linear foot of the cagebetween the feeder and choke ends of the cage.

In some embodiments, the method further comprises pelletizing the spentbiomass of reduced oil content or extruding the spent biomass of reducedoil content as a cake. In some embodiments, the method further comprisesextracting lipids from the spent biomass of reduced oil content. In somecases, the lipid content in the spent biomass of reduced oil content isat least 45 percent less than the lipid content of the microbial biomassbefore subjecting it to pressure. In some embodiments, the methodfurther comprises extracting lipids from the spent biomass of reducedoil content with an organic solvent. In some embodiments, the methodfurther comprises adjusting the moisture content of the microbialbiomass to between 0.1 and 5% before subjecting the microbial biomass topressure.

In some cases, the method comprises adjusting the moisture content ofthe microbial biomass to between 0.5% and 3.5% by weight beforesubjecting the microbial biomass to pressure. In some cases, the methodcomprises adjusting the moisture content of the microbial biomass tobetween 1.0% and 2.0% by weight before subjecting the microbial biomassto pressure. In some embodiments, the adjustment is achieved byconditioning the biomass with heat. In some cases, the conditioning withheat is performed using a vertical stacked conditioner.

In some embodiments, the method further comprises conditioning thebiomass to change its physical or physiochemical properties withoutreleasing more than 5% of the lipids to facilitate release of lipids ina subsequent step wherein the biomass is subjected to pressure. In somecases, the conditioning step comprises heating the biomass at 150-300°F. In some cases, the conditioning step comprises heating the biomass at200-270° F. In some cases, the conditioning step comprises heating thebiomass at 210-260° F. In some embodiments, the conditioning stepcomprises heating the biomass for a period of time between 20 and 60minutes. In some embodiments, the conditioning step comprises subjectingthe biomass to a first pressure that does not release more than 5% ofthe lipids in the biomass.

In some embodiments, the method further comprises treating the biomasswith an expander or extruder without releasing more than 5% of thelipids in the biomass before the step of subjecting the biomass topressure sufficient to release more than 5% of the lipids.

In some embodiments, the method further comprises adding a bulking agentto the microbial biomass to facilitate release of the lipids when themicrobial biomass is subjected to pressure. In some cases, the bulkingagent is selected from the group consisting of switchgrass, soybeanhulls, dried rosemary, corn stover, cellulose, spent biomass of reducedlipid content, and sugar cane bagasse. In some cases, the bulking agentis spent microbial biomass of reduced lipid content that comprisesbetween 40% and 90% polysaccharide and less than 10% oil. In some cases,the bulking agent is spent microbial biomass of reduced lipid contentthat comprises between 60% and 80% polysaccharide and less than 10% oil.In some cases, the bulking agent is spent microbial biomass of the samestrain as the microbial biomass. In some embodiments, the polysaccharideis of 20-30 mole percent galactose; 55-65% mole percent glucose; and5-15 mole percent mannose. In some cases, the spent biomass of reducedlipid content is from microalgae from the genus Chlorella, Parachlorellaor Prototheca. In some embodiments, the bulking agent has an averageparticle size of less than 1.5 mm In some cases, the bulking agent hasan average particle size of between 150-350 microns. In some cases, thebulking agent is added to the microbial biomass prior to a step ofdehydrating the microbial biomass to a moisture content of less than 6%.

In some embodiments of the present invention, the microbial biomass ismicroalgae. In some cases, the microalgae is selected from the specieslisted in Table 1. In some cases, the microalgae is of the genusChlorella, Parachlorella or Prototheca. In some embodiments, themicroalgae has a 23S rRNA genomic sequence with at least 75%, 85% or 95%nucleotide identity to one or more of SEQ ID NOs: 1-23 or 26-34.

In some embodiments of the present invention, the microbial biomass isbacteria. In some cases, the bacteria is from the genus Rhodococcus.

In some embodiments of the present invention, the microbial biomass isoleaginous yeast. In some cases, the oleaginous yeast is selected fromthe species listed in Table 2. In some cases, the oleaginous yeast isRhodotorula glutinis. In some embodiments, the oleaginous yeast has afungal 18S and 26S rRNA genomic sequence with at least 75%, 85%, or 95%nucleotide identity to one or more of SEQ ID NOs: 37-69. In someembodiments, the microbial biomass is an oleaginous yeast of the genusTorulaspora or Yarrowia.

In some embodiments of the present invention, the microbial biomass isnon-yeast oleaginous fungi. In some cases, the non-yeast oleaginousfungi is selected from the species listed in Table 3.

In some embodiments, the microbial biomass contains at least 45% lipidsby dry cell weight. In some cases, the microbial biomass has at least15% carbohydrate by dry weight. In some cases, the microbial biomass isderived from microalgae having a fatty acid profile of: less than 2%C14:0; about 13-16% C16:0; about 1-4% C18:0; about 64-71% C18:1; about10-15% C18:2; about 0.5-2% C18:3; and less than 1% carbon chain length20 or longer. In some cases, the microalgae has a fatty acid lipidprofile comprising of at least 15% C:16 fatty acids, at least 50% C18:1fatty acids, at least 7% C18:2 fatty acids, and less than 3% C10:0-C14:0fatty acids. In some cases, the microalgae has a fatty acid profile of:about 1-2% C14:0; about 16-26% C16:0; about 2-6% C18:0; about 58-68%C18:1; and about 7-11% C18:2. In some embodiments, the microalgae has alipid profile comprising at least 4% C8-C14 and contains an exogenousgene encoding a thioesterase with a preference for one or more fattyacid chain lengths of 8, 10, 12 and 14 carbon atoms. In some cases, themicroalgae has a lipid profile comprising between 10 and 40% C8-C14. Insome cases, the microbial biomass has a lipid profile comprising atleast 10% 16:1. In some embodiments, the microbial biomass contains atleast 30% lipids by weight. In some cases, the microbial biomasscontains at least 40% lipids by weight. In some cases, the microbialbiomass contains at least 50% lipids by weight. In some cases, themicrobial biomass contains between 60-70% lipids by weight.

In some embodiments, the extracted lipid has less than 0.01 milligram ofchlorophyll per kilogram of lipid. In some cases, the extracted lipidhas between 0.2 and 0.3 micrograms of carotenoids per milliliter oflipid.

In some embodiments, the microbial biomass contains an exogenous geneencoding a sucrose invertase.

In some embodiments, the microbial biomass has been subjected to apneumatic drying step prior to application of pressure.

In some embodiments, the extracted lipids comprise one or more of thefollowing: no more than 8 ppm chloride, no more than 2 ppm phosphorus,no more than 26 ppm potassium, no more than 12 ppm sodium, and no morethan 5 ppm sulfur.

In another aspect, the present invention is directed to a method ofpreparing hydrated microbial biomass for oil extraction. In oneembodiment, the method comprises adding a bulking agent to the biomass,and drying the bulking agent and the biomass together to a moisturecontent of less than 6%, thereby forming a dried bulking agent-biomassmixture. In some embodiments, the hydrated microbial biomass iscontained in a fermentation broth that has not been subjected toseparation or water removal processes. In some cases, the bulking agentis selected from the group consisting of switchgrass, soybean hulls,dried rosemary, corn stover, cellulose, spent biomass of reduced lipidcontent, and sugar cane bagasse. In some cases, the bulking agent isspent biomass of reduced lipid content that comprises between 40% and90% polysaccharide and less than 10% oil.

In yet another aspect, the present invention is directed to a method forextracting lipids from microbial biomass. In one embodiment, the methodcomprises (a) co-drying hydrated microbial biomass constituting at least20% lipids by weight and a bulking agent, therby forming a dried bulkingagent-biomass mixture, (b) conditioning the dried bulking agent-biomassmixture so that the moisture content is less than 4% by weight, and (c)subjecting the conditioned dried bulking agent-biomass mixture topressure, whereby cells of the biomass are lysed, releasing more than 5%of the lipids and leaving spent biomass of reduced lipid content. Insome embodiments, the hydrated microbial biomass is contained in afermentation broth that has not been subjected to separation or waterremoval processes. In some cases, the bulking agent is selected from thegroup consisting of switchgrass, soybean hulls, dried rosemary, cornstover, cellulose, spent biomass of reduced lipid content and sugar canebagasse. In some cases, the bulking agent is spent biomass of reducedlipid content that comprises between 40% and 90% polysaccharide and lessthan 10% oil.

In still another aspect, the present invention is directed to a methodof increasing yield in lipid extraction from microbial biomassconstituting at least 20% lipids by weight. In one embodiment, themethod comprises co-drying the microbial biomass with a bulking agent,whereby the amount of oil extracted from the co-dried microbial biomassand bulking agent when subjected to pressure is greater than without theaddition of the bulking agent. In some cases, the microbial biomass isderived from a culture that was cultivated through a process selectedfrom the group consisting of a heterotrophic process, a photoautotrophicprocess, and a mixotrophic process.

These and other aspects and embodiments of the invention are describedin the accompanying drawings, a brief description of which immediatelyfollows, and in the detailed description of the invention below, and areexemplified in the examples below. Any or all of the features discussedabove and throughout the application can be combined in variousembodiments of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1a shows microalgal biomass after the surface moisture has beenremoved through drum drying.

FIG. 1b shows microalgal biomass after being conditioned using a lowpressure “pre-press” to form collets.

FIG. 2a shows spent pressed cake from microbial biomass that is of poorquality for subsequent solvent extraction.

FIG. 2b shows spent pressed cake from microbial biomass that is of goodquality for subsequent solvent extraction.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods for extracting lipids frommicroorganisms. This detailed description of the invention is dividedinto sections for the convenience of the reader, beginning with sectionI, which provides definitions of various terms used in describing theinvention. Section II describes the methods of the invention forextracting oil from microorganisms, for preparing microbial biomass forthe extraction of oil, and for further processing spent biomass. SectionIII describes microorganisms useful in generating oil-containingmicrobial biomass and methods for culturing them to produce oil. SectionV provides illustrative examples of how to practice the methods of theinvention.

I. Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the meaning commonly understood by a person skilled in the art towhich this invention belongs. The following references provide one ofskill in the art to which this invention pertains with generaldefinitions of many of the terms used in this disclosure: Singleton etal., Dictionary of Microbiology and Molecular Biology (2^(nd) ed. 1994);The Cambridge Dictionary of Science and Technology (Walker ed., 1988);Glossary of Genetics, 5^(th) Ed., R. Rieger et al. (eds.), SpringerVerlag (1991); and Hale & Marham, The Harper Collins Dictionary ofBiology (1991). As used herein, the following terms have the meaningsascribed to them, unless specified otherwise.

“Area Percent” refers to the area of peaks observed using FAME GC/FIDdetection methods in which every fatty acid in the sample is convertedinto a fatty acid methyl ester (FAME) prior to detection. For example, aseparate peak is observed for a fatty acid of 14 carbon atoms with nounsaturation (C14:0) compared to any other fatty acid such as C14:1. Thepeak area for each class of FAME is directly proportional to its percentcomposition in the mixture and is calculated based on the sum of allpeaks present in the sample (i.e. [area under specific peak/total areaof all measured peaks]×100). When referring to lipid profiles of oilsand cells of the invention, “at least 4% C8-C14” means that at least 4%of the total fatty acids in the cell or in the extracted glycerolipidcomposition have a chain length that includes 8, 10, 12 or 14 carbonatoms.

“Axenic” refers to a culture of an organism that is free fromcontamination by other living organisms.

“Biomass” refers to material produced by growth and/or propagation ofcells. Biomass may contain cells and/or intracellular contents as wellas extracellular material. Extracellular material includes, but is notlimited to, compounds secreted by a cell.

“Bioreactor” refers to an enclosure or partial enclosure in which cells,e.g., microorganisms, are cultured, optionally in suspension.

“Bulking agent” and “press-aid” are used interchangeably herein andrefer to material that is suitable to add to feedstock (such as driedand/or conditioned biomass) to increase the fiber content of thefeedstock. Bulking agents include, but are not limited to, switchgrass,soybean hulls, spent biomass, and sugar cane bagasse. Bulking agentsfacilitate release of lipids (oil) from biomass, perhaps by increasingthe uniformity with which pressure can be applied to the component cellsof the biomass. In some cases, a press aid can also act as a filter aid,clarifying or reducing the amount of foots that is extracted with theoil. An example of a press aid that also acts as a filter aid iscellulose.

“Cellulosic material” means the products of digestion of cellulose,including glucose, xylose, disaccharides, oligosaccharides, lignin, andother molecules.

“Conditioned feedstock” is dried, oil-bearing microbial biomass that hasbeen physically altered in some way after being dried, typically withoutreleasing more than 5% of total oil from the biomass and heated to atemperature in the range of 70° C. to 150° C. (160° F. to 300° F.). Asused in this context, the substance to which “releasing” and “released”refer is oil. Examples of conditioning include putting the dried,oil-bearing microbial biomass through a vertical stacked conditioner, anexpander, an extruder, or an expeller; and/or subjecting the dried,oil-bearing microbial biomass to flaking, cracking, grinding, crushing,heating, steaming, thermal conditioning, low pressure, high pressure,and other methods for changing the physical nature of the dried,oil-bearing microbial biomass to maximize oil extraction usingnon-chemical or solventless extraction methods. Changes that occur uponsubjecting the dried, oil-bearing microbial biomass to conditioninginclude changes at the micron scale, such as ruptured cells walls, aswell as changes at macro scale, such as conversion of dried flakes intopellets without releasing oil by low pressure pressing.

“Delipidated meal” and “delipidated microbial biomass” refer tomicrobial biomass after oil (including lipid) has been extracted fromit, either through the use of an expeller press or solvent extraction orboth.

“Dry back” refers to the process of adding pressed cake (also referredto herein as spent biomass) back into the feed end of the press where itis mixed with unpressed biomass. Essentially, the pressed cake is actingas a bulking agent or press aid for the unpressed material. Residual oilin the pressed cake can be further recovered along with oil from theunpressed biomass using this process.

“Dry cell weight” refers the weight of microbial biomass once all orsubstantially all of the water (moisture) has been removed therefrom.

“Dry microbial biomass” refers to microbial biomass from which the freemoisture or surface moisture has been removed, usually so that themicrobial biomass contains less than 10%, and often less than 6%, ofmoisture by weight. In one embodiment, dried microbial biomass isderived from microalgae. In one embodiment, the dried microbial biomassis derived from microalgae that contains at least 20% lipids by dry cellweight after drying.

“Expander” refers to a low-shear extruder that heats, homogenizes,and/or shapes oilseeds and other oil-bearing material into porouscollets or pellets with a high bulk density. In one embodiment of anexpander-mediated process, steam is injected into oilseed flakes/cakesor oil-bearing material under pressure, and this mixture is extrudedthrough plates to the atmosphere. The collets expand when released tothe atmosphere, hence the name expander. Historically, the expander hasbeen used to prepare plant seed/oil seed derived collets for solventextraction because of the higher bulk density of the collets aftertreatment with the expander, which allows for more surface area andincreased efficiency in solvent extraction.

“Expeller press” means a screw press or continuous expeller that is usedfor mechanical extraction of oilseeds, such as but not limited tosoybeans and rapeseed/canola. Oil-bearing raw material (such asoilseeds) is fed into the machine at one end and the material issubjected to friction and high pressure from the screw drive that movesthe material along a shaft. Oil is released and seeps through smallopenings along the shaft and the solids (with reduced oil content) areexpelled at the end of the shaft as a pressed cake. Examples ofexpeller/screw presses include those that are marketed by AndersonInternational Corp. (Cleveland, Ohio), Alloco (Santa Fe, Argentina), DeSmet Rosedowns (Humberside, UK), The Dupps Co. (Germantown, Ohio), GrupoTecnal (Sao Paulo, Brazil), Insta Pro (Des Moines, Iowa), HarburgFreudenberger (previously Krupp Extraktionstechnik) (Hamburg, Germany),French Oil Mill Machinery Company (Piqua, Ohio), MaschinenfabrikReinartz (Neuss, Germany), Shann Consulting (New South Wales, Australia)and SKET (Magdeburg, Germany).

“Fiber” means the complex carbohydrates from plants and other fibercontaining sources such as microorganisms that cannot be digested byhumans. The complex carbohydrates found in fiber can include cellulose,hemicellulose and lignin, dextrins, pectins, beta-glucans andoligosaccharides.

“Fixed carbon source” refers to molecule(s) containing carbon, typicallyorganic molecules, that are present at ambient temperature and pressurein solid or liquid form during a fermentation.

“Hydrated microbial biomass” means microbial biomass containing at least10% moisture content that is in a liquid. In some embodiments, hydratedmicrobial biomass is contained in a fermentation broth that has not beensubjected to separation or water removal processes.

“Hydrocarbon” refers to: (a) a molecule containing only hydrogen andcarbon atoms, wherein the carbon atoms are covalently linked to form alinear, branched, cyclic or partially cyclic backbone to which thehydrogen atoms are attached; or (b) a molecule that primarily containshydrogen and carbon atoms that can be converted to contain only hydrogenand carbon atoms by one to four chemical reactions. Non-limitingexamples of the latter include hydrocarbons containing an oxygen atombetween one carbon and one hydrogen atom to form an alcohol molecule, aswell as aldehydes containing an oxygen atom. Methods for the reductionof alcohols to hydrocarbons containing only carbon and hydrogen atomsare well known. Another example of a hydrocarbon is an ester, in whichan organic group replaces a hydrogen atom (or more than one) in anoxygen acid. The molecular structure of hydrocarbon compounds variesfrom the simplest, in the form of methane (CH₄), which is a constituentof natural gas, to the very large and complex, such as as theasphaltenes found in crude oil, petroleum, and bitumens. Hydrocarbonsmay be in gaseous, liquid, or solid form, or any combination of theseforms, and may have one or more double or triple bonds between adjacentcarbon atoms in the backbone. Accordingly, the term includes linear,branched, cyclic or partially cyclic alkanes, alkenes, lipids, andparaffin. Examples include propane, butane, pentane, hexane, octane,squalene and carotenoids.

“Hydrogen:carbon ratio” refers to the ratio of hydrogen atoms to carbonatoms in a molecule on an atom-to-atom basis. The ratio may also be usedto refer to the number of carbon and hydrogen atoms in a hydrocarbonmolecule. For example, the hydrocarbon with the highest ratio ismethane, CH₄ (4:1).

“Hydrophobic fraction” refers to a portion, or fraction, of a materialthat is more soluble in a hydrophobic phase in comparison to an aqueousphase. A hydrophobic fraction is substantially insoluble in water andusually non-polar.

The phrase “increased lipid yield” refers to an increase in theproductivity of a microbial culture by, for example, increasing dryweight of cells per liter of culture, increasing the percentage of lipidin cells and/or the percentage of cells that constitute lipid, and/orincreasing the overall amount of lipid per culture volume per unit time.

The phrase “limiting concentration of a nutrient” refers to aconcentration of nutrient in a culture that limits the propagation of acultured organism. A “non-limiting concentration of a nutrient” is aconcentration that supports maximal propagation during a given cultureperiod. Thus, the number of cells produced during a given culture periodis lower in the presence of a limiting concentration of a nutrient thanwhen the nutrient is non-limiting. A nutrient is said to be “in excess”in a culture, when the nutrient is present at a concentration greaterthan that which supports maximal propagation.

“Lipid” refers to a lipophilic molecule from a biological organism.Biological functions of a lipid include, but are not limited to, storingenergy, serving as a structural component of a cell membrane, and actingas a signaling molecule. Lipid molecules are soluble in nonpolarsolvents (such as ether and chloroform) and are relatively or completelyinsoluble in water. Lipid molecules have these properties, because theyconsist largely of relatively long hydrocarbon chains which arehydrophobic in nature. Examples of lipids include fatty acids (saturatedand unsaturated); glycerides or glycerolipids (such as monoglycerides,diglycerides, triglycerides, and neutral fats, and phosphoglycerides orglycerophospholipids); nonglycerides (sphingolipids, sterol lipids,including cholesterol and steroid hormones, prenol lipids, includingterpenoids, waxes, and polyketides); and complex lipid derivatives(sugar-linked lipids, or glycolipids, and protein-linked lipids). Otherexamples of lipid include free fatty acids; esters of fatty acids;sterols; pigments (e.g., carotenoids and oxycarotenoids), phytosterols,ergothionine, lipoic acid, antioxidants including beta-carotene andtocopherol. Also included in the class of lipids are polyunsaturatedfatty acids such as arachidonic acid, stearidonic acid, cholesterol,desmesterol, astaxanthin, canthaxanthin, and n-6 and n-3 highlyunsaturated fatty acids such as eicosapentaenoic acid (EPA),docosapentaenoic acid, and docosahexaenoic acid (DHA). Microbial oil, asused herein, refers to lipid.

The phrase “lipid:organic solvent composition” refers to a mixture oflipid and organic solvent.

“Lysed” refers to having broken or disrupted the cellular or plasmamembrane and optionally the cell wall of a biological organism or cell,and releasing at least some intracellular content into the extracellularenvironment. “Lysis” refers to the breakage of the cellular or plasmamembrane and optionally the cell wall of a biological organismsufficient to release at least some intracellular content into theextracellular environment, often by mechanical, viral, osmotic, ortemperature variation mechanisms that compromise its integrity. “Lysing”refers to disrupting the cellular or plasma membrane and optionally thecell wall of a biological organism or cell sufficient to release atleast some intracellular content into the extracellular environment.

“Microalgae” refers to a microbial organism that contains a chloroplast,and optionally that is capable of performing photosynthesis. Microalgaeinclude obligate photoautotrophs, which cannot metabolize a fixed carbonsource as energy, as well as heterotrophs, which can live solely off ofa fixed carbon source. Microalgae can refer to unicellular organismsthat separate from sister cells shortly after cell division, such asChlamydomonas, and to microbes such as, for example, Volvox, which is asimple multicellular photosynthetic microbe of two distinct cell types.“Microalgae” can also refer to cells such as Chlorella and Dunaliella.“Microalgae” also includes other microbial photosynthetic organisms thatexhibit cell-cell adhesion, such as Agmenellum, Anabaena, andPyrobotrys. “Microalgae” also includes obligate heterotrophicmicroorganisms that have lost the ability to perform photosynthesis,such as certain dinoflagellate species and species of the genusPrototheca.

“Microbial biomass” refers to biomass derived from a microbe.

“Microorganism” and “microbe” are used interchangeably herein and referto microscopic unicellular organisms.

“Oil” refers to a hydrophobic, lipophilic, nonpolar carbon-containingsubstance including but not limited to geologically-derived crude oil,distillate fractions of geologically-derived crude oil, hydrocarbons,vegetable oil, algal oil, and microbial lipids.

“Oleaginous yeast” refers to a yeast that can accumulate more than 20%of its dry cell weight as lipid. Oleaginous yeast include organisms suchas Yarrowia lipolytica and other species of the Dikarya subkingdom offungi such as Rhodosporidium toruloides (Eukaryota; Fungi/Metazoa group;Fungi; Dikarya; Basidiomycota; Pucciniomycotina; Microbotryomycetes;Sporidiobolales; Rhodosporidium); Rhodotorula glutinis (Eukaryota;Fungi/Metazoa group; Fungi; Dikarya; Basidiomycota; Pucciniomycotina;Microbotryomycetes; Sporidiobolales; mitosporic Sporidiobolales;Rhodotorula); Lipomyces tetrasporus (Eukaryota; Fungi/Metazoa group;Fungi; Dikarya; Ascomycota; Saccharomyceta; Saccharomycotina;Saccharomycetes; Saccharomycetales; Lipomycetaceae; Lipomyces);Cryptococcus curvatus (Eukaryota; Fungi/Metazoa group; Fungi; Dikarya;Basidiomycota; Agaricomycotina; Tremellomycetes; Tremellales; mitosporicTremellales; Cryptococcus); Trichosporon domesticum (Eukaryota;Fungi/Metazoa group; Fungi; Dikarya; Basidiomycota; Agaricomycotina;Tremellomycetes; Tremellales; mitosporic Tremellales; Trichosporon);Yarrowia lipolytica (Eukaryota; Fungi/Metazoa group; Fungi; Dikarya;Ascomycota; Saccharomyceta; Saccharomycotina; Saccharomycetes;Saccharomycetales; Dipodascaceae; Yarrowia); Sporobolomycesalborubescens (Eukaryota; Fungi/Metazoa group; Fungi; Dikarya;Basidiomycota; Pucciniomycotina; Microbotryomycetes; Sporidiobolales;mitosporic Sporidiobolales; Sporobolomyces); Geotrichum vulgare(Eukaryota; Fungi/Metazoa group; Fungi; Dikarya; Ascomycota;Saccharomyceta; Saccharomycotina; Saccharomycetes; Saccharomycetales;Dipodascaceae; mitosporic Dipodascaceae; Geotrichum): and Torulasporadelbrueckii (Eukaryota; Fungi/Metazoa group; Fungi; Dikarya; Ascomycota;Saccharomyceta; Saccharomycotina; Saccharomycetes; Saccharomycetales;Saccharomycetaceae; Torulaspora). Within Dikarya, the invention includesuse of organisms from all sub-domains of Dikarya (Ascomycota andBasidiomycota) and taxonomic sub-classifications within Ascomycota andBasidiomycota.

“Organic solvent” refers to a carbon-containing material that dissolvesa solid, liquid, or gaseous solute, resulting in a solution.

“Photobioreactor” refers to a container, at least part of which is atleast partially transparent or partially open, thereby allowing light topass through, in which, e.g., one or more microalgae cells are cultured.Photobioreactors may be closed, as in the instance of a polyethylene bagor Erlenmeyer flask, or may be open to the environment, as in theinstance of an outdoor pond.

“Polysaccharide” (also called “glycan”) refers to carbohydrate made upof monosaccharides joined together by glycosidic linkages. Cellulose isan example of a polysaccharide that makes up certain plant cell walls.Cellulose can be depolymerized by enzymes to yield monosaccharides suchas xylose and glucose, as well as larger disaccharides andoligosaccharides. Other examples of polysaccharides include fiber,soluble and insoluble dietary fiber, hemicellulose, and the carbohydratefrom microbial cell walls, such as that contained in spent biomass.

“Polysaccharide-degrading enzyme” refers to any enzyme capable ofcatalyzing the hydrolysis, or depolymerization, of any polysaccharide.For example, cellulose catalyzes the hydrolysis of cellulose.

“Port”, in the context of a bioreactor, refers to an opening in thebioreactor that allows influx or efflux of materials such as gases,liquids and cells. Ports are usually connected to tubing leading fromthe photobioreactor.

“Pressing” refers to the application of sufficient pressure to forceintracellular oil from microbial biomass, which may also be referred toherein as a “pressing step.” Pressing may be sufficient to lyse all orsubstantially all of the cells in the microbial biomass.

“Spent biomass”, “spent microbial biomass” and “pressed cake” all referto microbial biomass that has been made into conditioned feedstock andthen has been subjected to high pressure so that the resulting materialhas less lipid content on a w/w basis than the conditioned feedstockfrom which it is derived. High pressure can be achieved by the use ofcompression pressure, such as that provided by machines such as anexpeller press, a screw oil expeller, and a mechanical press, as well asby direct hydraulic pressure and other processes so that the oil issqueezed out of the conditioned feed stock. In one embodiment, the spentmicrobial biomass is prepared by passing oil-bearing microbial biomassthrough an oilseed press. In one embodiment, the spent microbial biomassis microalgae biomass that has less than 30% oil by dry cell weight

“Suitable for animal feed” means a substance or material can be consumedwithout deleterious effect by an animal, typically a a non-human mammalof agricultural or veterinary interest, including but not limited tohorses, cattle, pigs, chickens, dogs and cats; in preferred embodiments,a material suitable for animal feed provides nutrition to the animal.

II. Methods for Extracting Oil from Microorganisms

In one aspect, the present invention provides methods for extracting,recovering, isolating, or otherwise obtaining oil (lipids) frommicroorganisms. The methods of the present invention are applicable toextracting a variety of lipids from a variety of microorganisms. In themethods of the present invention, the lipid-producing microorganism(e.g., a microalgae) is first cultivated under conditions that allowsfor lipid production to generate oil-containing microbial biomass. Theoil-containing biomass is then, depending on the method employed,optionally admixed with a bulking agent, and dried and conditioned toprepare a dry, conditioned feedstock that is then pressed to extract theoil. For the convenience of the reader, this discussion is divided intosubsections.

Subsection A describes the microbial biomass suitable for oil extractionin accordance with the methods of the invention. Subsection B describesmethods for removing water from the biomass, including dewatering anddrying. Subsection C describes methods for conditioning the biomass.Subsection D describes bulking agents (press aids) and their use withdry microbial biomass, hydrated microbial biomass, and conditionedfeedstock. Subsection E describes various methods for subjectingconditioned feedstock to pressure to extract oil (the pressing step).Subsection F describes the oil produced by the pressing step and methodsfor its use and further purification. Subsection G describes the spentbiomass of reduced oil content produced by the pressing step and methodsfor its use.

A. Suitable Biomass

While biomass from a wide variety of microbes, including microalgae,oleaginous bacteria, oleaginous yeast and fungi (see Section III,below), can be employed in the methods of the invention, microbialbiomass suitable for use in the methods described herein typicallycomprises at least 20% oil by dry cell weight. In some embodiments, thebiomass comprises oil in a range of from at least 25% to at least 60% ormore oil by dry cell weight. In some embodiments, the biomass containsfrom 15-90% oil, from 25-85% oil, from 40-80% oil, or from 50-75% oil bydry cell weight. In various embodiments of the invention, the microbialbiomass (dry or hydrated) or conditioned feedstock contains at least 25%oil by weight. In various embodiments, the dry microbial biomass orconditioned feedstock contains at least 25% lipids by weight or by drycell weight. In various embodiments, the dry microbial biomass orconditioned feedstock contains at least 40%, at least 50%, or at least75% lipids by weight or by dry cell weight. In various embodiments, thedry microbial biomass or conditioned feedstock contains at least 15%carbohydrate by weight or by dry cell weight.

The oil of the biomass described herein, or extracted from the biomassfor use in the methods and compositions of the present invention cancomprise glycerolipids with one or more distinct fatty acid ester sidechains. Glycerolipids are comprised of a glycerol molecule esterified toone, two, or three fatty acid molecules, which can be of varying lengthsand have varying degrees of saturation. The length and saturationcharacteristics of the fatty acid molecules (and thus the oil) can bemanipulated to modify the properties or proportions of the fatty acidmolecules in the oil of the present invention via culture conditions orvia lipid pathway engineering, as described herein (see also PCT PatentApplication Nos. US09/066,141 and US09/066,142, incorporated herein byreference). Thus, specific blends of algal oil can be prepared eitherwithin a single species of microalgae (or other microbe), or by mixingtogether the biomass or algal oil from two or more species of microalgae(or other microbe(s)).

The oil composition, i.e., the properties and proportions of the fattyacid constituents of the glycerolipids, can also be manipulated bycombining biomass or oil from at least two distinct genera or species ofmicrobes, i.e., microalgae. In some embodiments, at least two of thedistinct genera or species of microbes, i.e., microalgae, have differentglycerolipid profiles. The distinct species (or genera) of microbes canbe cultured together or separately as described herein (for microalgae,typically under heterotrophic conditions), to generate the respectiveoils. Different species of microbes can contain different percentages ofdistinct fatty acid constituents in the cell's glycerolipids.

In various embodiments, the microbial oil is primarily comprised ofmonounsaturated oil. In some cases, the oil is at least 50%monounsaturated oil by weight or volume. In various embodiments, the oilis at least 50%, at least 60%, at least 70%, or at least 80% or moremonounsaturated oil by weight or by volume. In some embodiments, the oilcomprises at least 10%, at least 20%, at least 30%, at least 40%, or atleast 50% or more esterified oleic acid or esterified alpha-linolenicacid by weight or by volume. In various embodiments, the oil comprisesless than 10%, less than 5%, or less than 1% by weight or by volume, oris substantially free of, esterified docosahexanoic acid (DHA).

In various embodiments of this and other aspects of the invention, thebiomass is prepared by fermentation of a microbe that contains 18:1fatty acid. In various embodiments, the microbe has a fatty acid profileof less than 1% C14:0; about 10-11% C16:0; about 3-4% C18:0; about70-71% C18:1; about 14-15% C18:2; about 1-2% C18:3; and less than 1%C20:0. In various embodiments, the microbe has a fatty acid profile ofabout 1-2% C14:0; about 20% C16:0; about 4% C18:0; about 64% C18:1; andabout 7-8% C18:2. In various embodiments, the microbe has at most 0.5%DHA. In these and other embodiments, the microbe is, in some instances,a microalgae.

Thus, a wide variety of microbial biomass is suitable for use in themethods of the invention. In accordance with these methods, theoil-containing biomass is typically dewatered, dried, conditioned, andthen pressed to extract the oil.

B. Dewatering and Drying the Microbial Biomass

The various embodiments of the methods of the invention involve one ormore steps of removing water (or other liquids) from the microbialbiomass. These steps of removing water can include the distinct stepsreferred to herein as dewatering and drying.

Dewatering, as used herein, refers to the separation of theoil-containing microbe from the fermentation broth (liquids) in which itwas cultured. Dewatering, if performed, should be performed by a methodthat does not result in, or results only in minimal loss in, oil contentof the biomass. Accordingly, care is generally taken to avoid cell lysisduring any dewatering step. Dewatering is a solid-liquid separation andinvolves the removal of liquids from solid material. Common processesfor dewatering include centrifugation, filtration, and/or the use ofmechanical pressure.

Centrifugation is a process that involves the use of centrifugal forcefor the separation of mixtures. The more dense components of the mixturemigrate away from the axis of the centrifuge, while the less densecomponents of the mixture migrate towards the axis. By increasing theeffective gravitational force (i.e., by increasing the centrifugationspeed), more dense material, usually solids, separate from the lessdense material, usually liquids, according to density.

Microbial biomass useful in the methods of the present invention can bedewatered from the fermentation broth through the use of centrifugation,to form a concentrated paste. After centrifugation, there is still asubstantial amount of surface or free moisture in the microbial biomass(e.g., upwards of 70%) and thus, centrifugation is not considered to be,for purposes of the present invention, a drying step. Optionally, aftercentrifugation, the biomass can be washed with a washing solution (e.g.,deionized water) to remove remaining fermentation broth and debris.

In some embodiments, dewatering involves the use of filtration. Oneexample of filtration that is suitable for the present invention istangential flow filtration (TFF), also known as cross-flow filtration.Tangential flow filtration is a separation technique that uses membranesystems and flow force to purify solids from liquids. For a preferredfiltration method see Geresh, Carb. Polym. 50; 183-189 (2002), whichdiscusses use of a MaxCell A/G technologies 0.45 uM hollow fiber filter.Also see for example Millipore Pellicon® devices, used with 100 kD, 300kD, 1000 kD (catalog number P2C01MC01), 0.1 uM (catalog numberP2VVPPV01), 0.22 uM (catalog number P2GVPPV01), and 0.45 uM membranes(catalog number P2HVMPV01). The retentate should not pass through thefilter at a significant level. The retentate also should not adheresignificantly to the filter material. TFF can also be performed usinghollow fiber filtration systems.

Non-limiting examples of tangential flow filtration include thoseinvolving the use of a filter with a pore size of at least about 0.1micrometer, at least about 0.12 micrometer, at least about 0.14micrometer, at least about 0.16 micrometer, at least about 0.18micrometer, at least about 0.2 micrometer, at least about 0.22micrometer, at least about 0.45 micrometer, or at least about 0.65micrometers. Preferred pore sizes of TFF allow solutes and debris in thefermentation broth to flow through, but not microbial cells.

In other embodiments, dewatering involves the use of mechanical pressuredirectly applied to the biomass to separate the liquid fermentationbroth from the microbial biomass. The amount of mechanical pressureapplied should not cause a significant percentage of the microbial cellsto rupture, if that would result in loss of oil, but should insteadsimply be enough to dewater the biomass to the level desired forsubsequent processing.

One non-limiting example of using mechanical pressure to dewatermicrobial biomass employs the belt filter press. A belt filter press isa dewatering device that applies mechanical pressure to a slurry (e.g.,microbial biomass that is directly from the fermentor or bioreactor)that is passed between the two tensioned belts through a serpentine ofdecreasing diameter rolls. The belt filter press can actually be dividedinto three zones: gravity zone, where free draining water/liquid isdrained by gravity through a porous belt; a wedge zone, where the solidsare prepared for pressure application; and a pressure zone, whereadjustable pressure is applied to the gravity drained solids.

One or more of the above dewatering techniques can be used alone or incombination to dewater the microbial biomass for use in the presentinvention. The present invention results in part from the discovery thatthe moisture content of the microbial biomass (conditioned feedstock)dramatically affects the yield of oil obtained in the pressing step, andthat the optimal moisture level, below 6% and preferably below 2%, isquite different from the optimal moisture levels for pressing oil frommany oil-bearing seeds. While the optimal moisture level can varydepending on the type of oil-bearing seed, and can also vary dependingon the type of microbial biomass, the optimal moisture level forpressing microbial biomass is less than that for oil seeds. For example,the optimal moisture content for pressing sesame and linseed is about 4%(Willems et al., J. Food Engineering 89:1, pp. 8-16, 2008). The optimalmoisture content for pressing crambe seeds is between 9.2 and 3.6%(Singh et al., JAOCS 79:2, pp. 165-170, 2006). The optimal moisturecontent for pressing canola seeds is about 5% (Vadke et al., JAOCS 65:7,pp. 1169-1176, 1988). The optimal moisture content for pressing coconutis about 11% (Mpagalile et al., Int. J. Food Sciences and Nutrition,56:2, pp. 125-132, 2005). Other optimal moisture contents are 7% forrapeseed, 6% for camelina, 8.5% for sunflower, 11% for safflower and 12%for soybean (Alam, M. S. November 2007. Basics of Fats and OilsChemistry: Factors Affecting Crude Oil Quality. Presented to theVegetable Oils Extraction Short Course, Texas A&M Food Protein R&DCenter, College Station, Tex.).

In contrast, the optimal moisture content for pressing microbial biomassis less than 6% by weight, and more preferably less than 3%. Forexample, optimal moisture content can be 0.5-2% by weight. In variousembodiments, particularly those relating to the extraction of oil frommicroalgal biomass, the optimal moisture content is in the range of 0.5%to 2% of the total weight of the microbial biomass. In one embodiment,the moisture content is in the range of 0.7% to 1.2% of the total weightof the microbial biomass. In one embodiment, the moisture content is inthe range of 1.0% to 2.0% of the total weight of the microbial biomass.The optimal moisture level can depend on several factors, including butnot limited to the percent lipids (oil) as measured by dry cell weight(DCW) or the amount of fiber and hemicellulose in the biomass. In someembodiments of the methods of the invention, such as, for example, thosein which a bulking agent is employed (see subsection D), dewateringalone provides a suitable moisture content of the microbial biomass thatis then conditioned prior to the pressing step. In other methods andembodiments of the invention, dewatered biomass is subjected to a dryingstep and then conditioned prior to the pressing step (in which oil isextracted from the biomass).

Drying, as referred to herein, refers to the removal of some or all ofthe free moisture or surface moisture of the microbial biomass. Likedewatering, the drying process should not result in significant loss ofoil from the microbial biomass. Thus, the drying step should typicallynot cause lysis of a significant number of the microbial cells, becausein most cases, the lipids are located in intracellular compartments ofthe microbial biomass. Several methods of drying microbial biomass knownin the art for other purposes are suitable for use in the methods of thepresent invention. Microbial biomass after the free moisture or surfacemoisture has been removed is referred to as dried microbial biomass. Ifno further moisture removal occurs in the conditioning or moisturereduction occurs via the addition of a dry bulking agent prior to thepressing step, then the dried microbial biomass should contain less than6% moisture by weight.

In various embodiments, the dry microbial biomass has a moisture contentin the range of 0.1% to 5% by weight. In various embodiments, the drymicrobial biomass has a moisture content of less than 4% by weight. Invarious embodiments, the dry microbial biomass has a moisture content inthe range of 0.5% to 3.5% by weight. In various embodiments, the drymicrobial biomass has a moisture content in the range of 0.1% to 3% byweight. Non-limiting examples of drying methods suitable for use inpreparing dry microbial biomass in accordance with the methods of theinvention include lyophilization and the use of dryers such as a drumdryer, spray dryer, and a tray dryer, each of which is described below.

Lyophilization, also known as freeze drying or cryodessication, is adehydration process that is typically used to preserve a perishablematerial. The lyophilization process involves the freezing of thematerial and then reducing the surrounding pressure and adding enoughheat to allow the frozen water in the material to sublime from the solidphase to gas. In the case of lyophilizing microbial biomass, such asmicroalgae derived biomass, the cell wall of the microalgae acts as acryoprotectant that prevents degradation of the intracellular lipidsduring the freeze dry process.

Drum dryers are one of the most economical methods for drying largeamounts of microbial biomass. Drum dryers, or roller dryers, consist oftwo large steel cylinders that turn toward each other and are heatedfrom the inside by steam. In some embodiments, the microbial biomass isapplied to the outside of the large cylinders in thin sheets. Throughthe heat from the steam, the microbial biomass is then dried, typicallyin less than one revolution of the large cylinders, and the resultingdry microbial biomass is scraped off of the cylinders by a steel blade.The resulting dry microbial biomass has a flaky consistency. In variousembodiments, the microbial biomass is first dewatered and then driedusing a drum dryer. More detailed description of a drum dryer can befound in U.S. Pat. No. 5,729,910, which discloses a rotary drying drum.

Spray drying is a commonly used method of drying a liquid feed using ahot gas. A spray dryer takes a liquid stream (e.g., containing themicrobial biomass) and separates the solute as a solid and the liquidinto a vapor. The liquid input stream is sprayed through a nozzle into ahot vapor stream and vaporized. Solids form as moisture quickly leavesthe droplets. The nozzle of the spray dryer is adjustable, and typicallyis adjusted to make the droplets as small as possible to maximize heattransfer and the rate of water vaporization. The resulting dry solidsmay have a fine, powdery consistency, depending on the size of thenozzle used. In other embodiments, spray dryers can use a lyophilizationprocess instead of steam heating to dry the material.

Tray dryers are typically used for laboratory work and small pilot scaledrying operations. Tray dryers work on the basis of convection heatingand evaporation. Fermentation broth containing the microbial biomass canbe dried effectively from a wide range of cell concentrations using heatand an air vent to remove evaporated water.

Flash dryers are typically used for drying solids that have beende-watered or inherently have a low moisture content. Also known as“pneumatic dryers”, these dryers typically disperse wet material into astream of heated air (or gas) which conveys it through a drying duct.The heat from the airstream (or gas stream) dries the material as it isconveyed through the drying duct. The dried product is then separatedusing cyclones and/or bag filters. Elevated drying temperatures can beused with many products, because the flashing off of surface moistureinstantly cools the drying gas/air without appreciably increasing theproduct temperature. More detailed descriptions of flash dryers andpneumatic dryers can be found in U.S. Pat. No. 4,214,375, whichdescribes a flash dryer, and U.S. Pat. Nos. 3,789,513 and 4,101,264,which describe pneumatic dryers.

Regardless of the method selected for a drying step, the objective ofthe drying step is to reduce moisture content in the microbial biomass.If moisture is not removed from the dry microbial biomass during theconditioning step or reduced via the addition of a dry bulking agent,then the moisture content should be less than 6% by weight. Typically,the dry microbial biomass (conditioned feedstock) suitable for pressinghas a moisture content of about 0.1% to 6% by weight, including invarious embodiments, a moisture content of 0.5-2.5%. Moisture may beadded back to the biomass, if necessary, after drying to adjust moisturecontent to the optimal level. If the dry microbial biomass will beadmixed with a dry bulking agent (see subsection D) or conditioned in amanner that will reduce moisture content further (see subsection C),then higher (above 6% by weight) moisture content may be acceptable, asbulking agents and/or conditioning can, in some embodiments, reduce themoisture content to the desired optimal level.

Dewatered and/or dried microbial biomass is conditioned prior to thepressing step, as described in the following subsection.

C. Conditioning the Microbial Biomass

Conditioning of the microbial biomass helps to achieve desired levels ofoil extraction. Conditioning refers to heating the biomass to atemperature in the range of 70° C. to 150° C. (160° F. to 300° F.) andchanging the physical or physiochemical nature of the microbial biomassto improve oil yields in the subsequent oil extraction (pressing) step.Conditioning microbial biomass results in the production of “conditionedfeedstock.” In addition to heating or “cooking” the biomass,non-limiting examples of conditioning the biomass include adjusting themoisture content within the dry microbial biomass, subjecting the drymicrobial biomass to a low pressure “pre-press”, subjecting the drymicrobial biomass to cycles of heating and cooling, subjecting the drymicrobial biomass to an expander, and/or adjusting the particle size ofthe dry microbial biomass.

The conditioning step can include techniques (e.g., heating orapplication or pressure) that overlap in part with techniques used inthe drying or pressing steps. However, the primary goals of these stepsare different: the primary goal of the drying step is the removal ofsome or all of the free moisture or surface moisture from the microbialbiomass. The primary goal of the conditioning step is to heat thebiomass, which can optionally result in the removal of intracellularwater from, i.e., adjusting the intracellular moisture content of, themicrobial biomass and/or altering the physical or physiochemical natureof the microbial biomass without substantial release of lipids tofacilitate release of oil during the pressing step. The primary the goalof the pressing step is to release oil from the microbial biomass orconditioned feedstock, i.e., the extraction of the oil.

In various embodiments, conditioning involves altering, or adjusting,the moisture content of the microbial biomass by the application ofheat, i.e., heat conditioning. Heat conditioning, as used herein, refersto heat treatment (either direct or indirect) of microbial biomass. Themoisture content of the microbial biomass can be adjusted byconditioning using heat (either direct or indirect), which is typicallydone, if at all, after a drying step. Even though the biomass may bedried by any of the above described methods, the moisture content of themicrobial biomass after drying can range, for example, from 3% to 15%moisture by weight, or 5-10% moisture by weight. Such a moisture rangemay not be optimal for maximal oil recovery in the pressing step.Therefore, there may be benefit in heat-conditioning dewatered and/ordry microbial biomass to adjust the moisture level to a level (below 6%)optimal for maximal oil recovery.

Heat conditioners used in oil seed processing are suitable for use inconditioning microbial biomass in accordance with the methods of thepresent invention, such as vertical stacked conditioners. These consistof a series of three to seven or moreclosed, superimposed cylindricalsteel pans. Each pan is independently jacketed for steam heating on bothsides and bottom and is equipped with a sweep-type stirrer mounted closeto the bottom, and operated by a common shaft extending through theentire series of pans. The temperature of the heat conditioner is alsoadjustable through regulation of the steam heating. There is anautomatically operated gate in the bottom of each pan, except the last,for discharging the contents to the pan below. The top pan is providedwith spray jets for the addition of moisture if desired. While moistureis sprayed onto seeds in many agricultural oil extraction processesduring conditioning, this common process is not desirable forconditioning microbial biomass. Cookers also typically have an exhaustpipe and fan for removal of moisture. Thus, it is possible to controlthe moisture of the microbial biomass, not only with respect to finalmoisture content but also at each stage of the operation. In thisrespect, a conditioning step of heating microbial biomass for anextended period of time (10-60 minutes for example) provides the effectof not only reducing moisture and increasing the temperature of thebiomass, but also altering the biophysical nature of the microbialbiomass beyond any heating effects that might occur in a subsequentpressing step, i.e., simply from friction of the material as it isforced through, e.g., a press.

Additionally, a steam jacketed horizontal cooker is another type of heatconditioner that is suitable for use in accordance with the methods ofthe invention herein. In this design, the biomass is mixed, heated andconveyed in a horizontal plane in deeper beds as compared toconventional vertical stacked cookers. In the horizontal cooker, theaction of a specially designed auger mixes conveys the biomass, whilethe biomass is simultaneously heated with indirect steam from the steamjacket. Water and vapor and air are vented out from the cooker throughan upper duct, which may or may not have an exhaust fan depending on thecooker's capacity. For cooking biomass at a high flow rate, severalhorizontal cookers can be stacked together. In this configuration, thebiomass is fed into the top level cooker and heated and conveyed throughby the auger and then thrown by gravity into a lower level cooker wherethe process is repeated. Several levels of horizontal cookers can bestacked together depending on the needed flow rate and thetime/temperature of conditioning required. Moisture and temperature canbe monitored and adjusted independently for each horizontal cookerlevel.

For the heat conditioning of microbial biomass, especially microalgalbiomass, the optimal time and temperature that the biomass spends in avertical stacked conditioner can vary depending on the moisture level ofthe biomass after drying. Heat conditioning (sometimes referred to as“cooking”) should not result in burning or scorching significant amountsof the microbial biomass during cooking. Depending on the moisturecontent of the microbial biomass prior to heat conditioning, i.e., forvery low levels of moisture, it may be beneficial or even necessary tomoisten the biomass before heat conditioning to avoid burning orscorching. Depending on the type of microbial biomass that is going tobe fed through an expeller press, the optimal temperature for heatconditioning will vary. For some types of microalgal, the optimaltemperature for heat conditioning is between 200-270° F. In someembodiments, the microalgal biomass is heat conditioned at 210-230° F.In other embodiments, the microalgal biomass is heat conditioned at220-270° F. In still other embodiments, the microalgal biomass is heatconditioned at 240-260° F. These temperature ranges are, like moisturecontent, significantly different from what is typically used in anoilseed conditioning process, as oilseed processes typically use lowerconditioning temperatures.

Heating the oil-bearing microbial biomass before pressing can aid in theliberation of oil from and/or accessing the oil-laden compartments ofthe cells. Oil-bearing microbial biomass contains the oil incompartments made of cellular components such as proteins andphospholipids. Repetitive cycles of heating and cooling can denature theproteins and alter the chemical structure of the cellular components ofthese oil compartments and thereby provide better access to the oilduring the subsequent extraction process. Thus, in various embodimentsof the invention, the microbial biomass is conditioned to prepareconditioned feedstock that is used in the pressing step, and theconditioning step involves heating and, optionally, one or more cyclesof heating and cooling.

If no further heat conditioning or other conditioning that altersmoisture content is to be performed, and if no bulking agent that willalter moisture content is to be added, then the conditioned feedstockresulting from heat conditioning should contain less than 6% moisture byweight. In various embodiments, the conditioned feedstock has a moisturecontent in the range of 0.1% to 5% by weight. In various embodiments,the conditioned feedstock has a moisture content of less than 4% byweight. In various embodiments, the conditioned feedstock has a moisturecontent in the range of 0.5% to 3.5% by weight. In various embodiments,the conditioned feedstock has a moisture content in the range of 0.1% to3% by weight.

In addition to heating the biomass, conditioning can, in someembodiments, involve the application of pressure to the microbialbiomass. To distinguish this type of conditioning from the pressureapplied during oil extraction (the pressing step), this type ofconditioning is referred to as a “pre-press.” The pre-press is conductedat low pressure, a pressure lower than that used for oil extraction inthe pressing step. Ordinary high-pressure expeller (screw) presses maybe operated at low pressure for this pre-press conditioning step.Pre-pressing the biomass at low pressure may aid in breaking open thecells to allow for better flow of oil during the subsequent highpressure pressing; however, pre-pressing does not cause a significantamount (e.g. more than 5%) of the oil to separate from the microbialbiomass. Also, the friction and heat generated during the pre-press mayalso help break open the oil compartments in the cells. Pre-pressing thebiomass at low pressure also changes the texture and particle size ofthe biomass, because the biomass will extrude out of the press in apellet-like form. In some embodiments, an extruder (see discussionbelow) is used to achieve the same or similar results as a low pressurepre-press conditioning step. In some embodiments, the pellets ofconditioned biomass are further processed to achieve an optimal particlesize for the subsequent full pressure pressing.

Thus, another parameter relevant to optimal extraction of oil frommicrobial biomass is the particle size. Typically, the optimum particlesize for an oil expeller press (screw press) is approximately 1/16^(th)of an inch thick. Factors that may affect the range of particle sizeinclude, but are not limited to, the method used to dry the microbialbiomass and/or the addition of a bulking agent or press aid to thebiomass. If the biomass is tray dried, e.g., spread wet onto a tray andthen dried in an oven, the resulting dried microbial biomass may need tobe broken up into uniform pieces of the optimal particle size to make itoptimal for pressing in an expeller press. The same is true if a bulkingagent is added to the microbial biomass before the drying process. Thus,conditioning may involve a step that results in altering the particlesize or average particle size of the microbial biomass. Machines such ashammer mills or flakers may be employed in accordance with the methodsof the invention to adjust the thickness and particle size of theoil-bearing microbial biomass.

In similar fashion, improved oil extraction can result from alteringother physical properties of the dried microbial biomass. In particular,the porosity and/or the density of the microbial biomass can affect oilextraction yields. In various embodiments of the methods of theinvention, conditioning of the biomass to alter its porosity and/ordensity is performed. Commonly used prior to hexane or other solventextraction of oil from oil seeds, expanders and extruders increase theporosity and the bulk density of the feedstock. In accordance with themethods of the present invention, expanders and extruders can beemployed to condition the microbial biomass before oil extraction andmay or may not cause a significant amount of oil to separate from themicrobial biomass. Both expanders and extruders are low-shear machinesthat heat, homogenize, and shape oil-bearing material into collets orpellets. Expanders and extruders work similarly; both have a worm/collarsetup inside a shaft such that, as it moves the material inside theshaft, mechanical pressure and shearing break open the cells. Thebiggest difference between expanders and extruders is that the expanderuses water and/or steam to puff the material at the end of the shaft.The sudden high pressure (and change in pressure) causes the moisture inthe material to vaporize, thus “puffing” or expanding the material usingthe internal moisture. Extruders change the shape of the material,forming collets or pellets. Extruders also lyse the cells and vaporizeswater from the biomass (reduction of moisture) while increasing thetemperature of the biomass (heating the biomass) through mechanicalfriction that the extruder exerts on the biomass. Thus, extruders andexpanders can be used in accordance with the methods of the invention tocondition the dry microbial biomass. The extruder/expanders can breakopen the cells, freeing the intracellular lipids, and can also changethe porosity and the bulk density of the material. These changes in thephysical properties of the feedstock may be advantageous in subsequentoil extraction.

The above-described conditioning methods can be used alone or incombination in accordance with the methods of the invention to achievethe optimal conditioned microbial biomass feedstock for subsequent oilextraction. Thus, the conditioning step involves the application of heatand optionally pressure to the biomass. In various embodiments, theconditioning step comprises heating the biomass at a temperature in therange of 70° C. to 150° C. (160° F. to 300° F.). In various embodiments,the heating is performed using a vertical stacked shaker. In variousembodiments, the conditioning step further comprises treating the drybiomass with an expander or extruder to shape and/or homogenize thebiomass.

D. Bulking Agents (Press Aids)

In various embodiments of the invention, a bulking agent or press aid isadded to the microbial biomass, which may be either dry or hydrated(i.e., biomass that has not been dried or that contains significant,i.e., more than 6% by weight, moisture, including biomass infermentation broth that has not been subjected to any process to removeor separate water) microbial biomass or conditioned feedstock, prior tothe pressing step. In various embodiments, the bulking agent has anaverage particle size of less than 1.5 mm In some embodiments, thebulking agent or press aid has a particle size of between 50 microns and1.5 mm. In other embodiments, the press aid has a particle size ofbetween 150 microns and 350 microns. In some embodiments, the bulkingagent is a filter aid. In various embodiments, the bulking agent isselected from the group consisting of cellulose, corn stover, driedrosemary, soybean hulls, spent biomass (biomass of reduced lipid contentrelative to the biomass from which it was prepared), including spentmicrobial biomass, sugar cane bagasse, and switchgrass. In variousembodiments, the bulking agent is spent microbial biomass (seesubsection G below) that contains between 40% and 90% polysaccharide byweight, such as cellulose, hemicellulose, soluble and insoluble fiber,and combinations of these different polysaccharides and/or less than 10%oil by weight. In various embodiments, the polysaccharide in the spentmicrobial biomass used as a bulking agent contains 20-30 mole percentgalactose, 55-65 mole percent glucose, and/or 5-15 mole percent mannose.

Thus, the addition of a press aid or bulking agent may be advantageousin some embodiments of the invention. When there is high oil content andlow fiber in the biomass, feeding the biomass through a press can resultin an emulsion. This results in low oil yields, because the oil istrapped within the solids. One way in accordance with the methods of theinvention to improve the yield in such instances is to addpolysaccharide to the biomass in the form of a bulking agent, also knownas a “press aid” or “pressing aid”. Bulking agents are typically highfiber additives that work by adjusting the total fiber content of themicrobial biomass to an optimal range. Microbial biomass such asmicroalgae and the like typically have very little crude fiber content.Typically, microbial biomass including microalgae biomass have a crudefiber content of less than 2%. The addition of high fiber additives (inthe form of a press aid) may help adjust the total fiber content of themicrobial biomass to an optimal range for oil extraction using anexpeller press. Optimal fiber content for a typical oil seed may rangefrom 10-20%. In accordance with the methods of the present invention, itmay be helpful to adjust the fiber content of the microbial biomass foroptimal oil extraction. The range for fiber content in the biomass maybe the same or a similar range as the optimal fiber content for atypical oil seed, although the optimal fiber content for each microbialbiomass may be lower or higher than the optimal fiber content of atypical oil seed. Suitable pressing aids include, but are not limitedto, switchgrass, rice straw, sugar beet pulp, sugar cane bagasse,soybean hulls, dry rosemary, cellulose, corn stover, delipidated (eitherpressed or solvent extracted) cake from soybean, canola, cottonseed,sunflower, jatropha seeds, paper pulp, waste paper and the like. In someembodiments, the spent microbial biomass of reduced lipid content from aprevious press is used as a bulking agent. In some applications,especially when the oil is going to be used in a food application or isgoing to be consumed, the pressing aid used in mixing with the microbialbiomass (dry or hydrated) or conditioned feedstock will be selected tomeet regulatory requirements (for use as a foodstuff). Thus, bulkingagents, when incorporated into a biomass, change the physiochemicalproperties of the biomass so as to facilitate more uniform applicationof pressure to cells in the biomass.

In some cases, the bulking agent can be added to the microbial biomassafter it has been dried, but not yet conditioned. In such cases, it mayadvantageous to mix the dry microbial biomass with the desired amount ofthe press aid and then condition the microbial biomass and the press aidtogether before feeding to a screw press. In other cases, the press aidcan be added to a hydrated microbial biomass before the microbialbiomass has been subjected to any separation or dewatering processes,drying, or conditioning. In such cases, the press aid can be addeddirectly to the fermentation broth containing the microbial biomassbefore any dewatering or other step.

The invention provides various methods relating to the extraction of oilfrom microbial biomass that employ the bulking agents described above.In one method, hydrated microbial biomass suitable for oil extraction isprepared by adding a bulking agent to the biomass and drying the mixtureobtained thereby to a moisture content less than 6% by weight, therebyforming a dried bulking agent/biomass mixture. In another method, oil isextracted from microbial biomass by co-drying hydrated microbial biomasscontaining at least 20% oil (including at least 40% oil) by weight and abulking agent to form a dried bulking agent/biomass mixture; reducingthe moisture content in the mixture to less than 4% by weight, i.e., bydrying and/or conditioning; and pressing the reduced moisture contentmixture to extract oil therefrom, thereby forming spent biomass ofreduced lipid content. In another method, increased yields of oil areobtained from microbial biomass containing at least 20% lipid by weightby co-drying the microbial biomass with a bulking agent, because theco-dried mixture will, upon pressing, release more oil than can beobtained from the biomass under the same conditions in the absence of abulking agent. In various embodiments of these and other methods of theinvention, the hydrated microbial biomass is contained in fermentationbroth that has not been subjected to processes to separate or removewater from the biomass.

In an embodiment, the bulking agent is spent microbial biomass,optionally that has been processed or milled (for homogeneous and easeof blending), that is combined with microbial biomass that has not beenextracted. In such cases, the total polysaccharide content of theblended (spent biomass as a press aid and non-extracted microbialbiomass) microbial biomass before it is fed into an expeller presscontains between 10% and 40% of the total weight of the blended biomass.

E. Pressing Microbial Biomass

Thus, in accordance with the methods of the invention conditionedfeedstock, optionally comprising a bulking agent, is subjected topressure in a pressing step to extract oil, producing oil separated fromthe spent biomass. The pressing step involves subjecting pressuresufficient to extract oil from the conditioned feedstock. Thus, in someembodiments, the conditioned feedstock that is pressed in the pressingstep comprises oil predominantly or completely encapsulated in cells ofthe biomass. In other embodiments, the biomass comprises predominantlylysed cells and the oil is thus primarily not encapsulated in cells.

In various embodiments of the different aspects of the invention, thepressing step will involve subjecting the conditioned feedstock to atleast 10,000 psi of pressure. In various embodiments, the pressing stepinvolves the application of pressure for a first period of time and thenapplication of a higher pressure for a second period of time. Thisprocess may be repeated one or more times (“oscillating pressure”). Invarious embodiments, more than 5 cycles of oscillating pressure areapplied. In various embodiments, one or more of the subsequent cyclesmay exert an average pressure that is higher than the average pressureexerted in one or more earlier cycles. For example and withoutlimitation, the average pressure in the last cycle can be at least2-fold higher than the average pressure in the first or any earliercycle. In various embodiments, moisture content of conditioned feedstockis controlled during the pressing step. In various embodiments, themoisture is controlled in a range of from 0.1% to 3% by weight.

In various embodiments, the pressing step is conducted with an expellerpress. In various embodiments, the pressing step is conducted in acontinuous flow mode. In various embodiments, the oiling rate is atleast 500 g/min to no more than 1000 g/min. In various continuous flowembodiments, the expeller press is a device comprising a continuouslyrotating worm shaft within a cage having a feeder at one end and a chokeat the opposite end, having openings within the cage is utilized. Theconditioned feedstock enters the cage through the feeder, and rotationof the worm shaft advances the feedstock along the cage and appliespressure to the feedstock disposed between the cage and the choke, thepressure releasing oil through the openings of cage and extruding spentbiomass from the choke end of the cage. In various embodiments, the cagehas an internal length that is between at least ten times to at least 20times its internal diameter. In various embodiments, the cage comprisesa plurality of elongated bars with at least some of the elongated barsseparated by one or more spacers, the bars resting on a frame, whereinthe one or more spacers between the bars form the openings, and oil isreleased through the openings to a collecting vessel fluidly coupledwith the cage. In various embodiments, the spacers between the elongatedbars are of different thicknesses thereby allowing variation of thespace between each elongated bar. In various embodiments, either thespacers or the gaps between the bars are from 0.005 to 0.030 inchesthick.

The cage on some expeller press can be heated using steam or cooledusing water depending on the optimal temperature needed for maximumyield. Optimal temperature should be enough heat to aid in pressing, butnot too high heat as to burn the biomass while it feeds through thepress. The optimal temperature for the cage of the expeller press canvary depending on the microbial biomass that is to be pressed. In someembodiments, for pressing microbial or microalgal biomass, the cage ispreheated and held to a temperature of between 200-270° F. In otherembodiments, the optimal cage temperature for microbial or some speciesof microalgal biomass is between 210-230° F. In still other embodiments,the optimal cage temperature for microbial or some species of microalgalbiomass is between 240-260° F. These temperature ranges differsignificantly from many oilseed pressing processes, and in fact someoilseed pressing processes are referred to as “cold pressing” due to thelack of heating the seeds or the press during the process.

In various embodiments, the pressure increases by a factor of between 10and 20 from the feeder end to the choke end of the cage. In variousembodiments, the pressure along the cage does not increase by more than100% of the pressure at the feeder end of the cage per linear foot ofthe cage between the feeder and choke ends of the cage. In variousembodiments, the power consumed by the device does not increase by morethan 10% when fully loaded with biomass or conditioned feedstockrelative to running empty. In various embodiments, the residence time offeedstock in the barrel of the device is no longer than 5-10 min. Invarious embodiments, either the temperature of the device or thepressure exerted by the device or both are monitored and/or controlled.

In various embodiments, pressure is controlled by adjusting rotationalvelocity of a worm shaft. In various embodiments, including those inwhich pressure is not controlled, an expeller (screw) press comprising aworm shaft and a barrel can be used. In various embodiments, the barrelhas a length and a channel having a diameter sized to receive the wormshaft, and wherein the barrel length is at least 10 to 15 times greaterthan the channel diameter. In various embodiments, the barrel of thepress has an entrance and an exit and the diameter of the worm shaftincreases from the entrance to the exit, and the pressing comprisesincreasing the pressure from the entrance to the exit of the barrel; invarious embodiments, the pressure at the exit is 12 to 16 or even up to20 times higher than the pressure at the entrance. In variousembodiments, the expeller (screw) press comprises a worm shaft and abarrel having a first channel and a second channel, both channelsconcentric and sized to receive the worm shaft, wherein the firstchannel has a first diameter and the second channel has a seconddiameter different than the first diameter. In various embodiments, theconditioned feedstock remains resident in the barrel of the screw pressfor 5 to 10 minutes.

In various embodiments, the expeller (screw) press comprises a wormshaft disposed in a barrel lined with a plurality of elongate barsseparated by one or more spacers therebetween, the spacers creating agap between the elongate bars. In such a press, pressure can becontrolled by adjusting the gap by changing the size or number ofspacers between the elongate bars, and/or if the press has a spacebetween an outer surface of the worm shaft and an inner surface of theelongate bars, pressure can be controlled by replacing at least some ofthe elongate bars with different sized bars so as to change the space.In various embodiments, the press comprises an output aperture and anadjustable choke coupled therewith, and pressure is controlled byadjusting the choke to increase or decrease the pressure. In variousembodiments, the expeller (screw) press comprises a worm shaft disposedin a barrel, and pressure is controlled by adjusting a gap between anouter surface of the worm shaft and an inside surface of the barrel.

Expeller presses (screw presses) are routinely used for mechanicalextraction of oil from soybeans and oil seeds. Generally, the mainsections of an expeller press include an intake, a rotating feederscrew, a cage or barrel, a worm shaft and an oil pan. The expeller pressis a continuous cage press, in which pressure is developed by acontinuously rotating worm shaft. An extremely high pressure,approximately 10,000-20,000 pounds per square inch, is built up in thecage or barrel through the action of the worm working against anadjustable choke, which constricts the discharge of the pressed cake(spent biomass) from the end of the barrel. In various embodiments,screw presses from the following manufacturers are suitable for use :Anderson International Corp. (Cleveland, Ohio), Alloco (Santa Fe,Argentina), De Smet Rosedowns (Humberside, UK), The Dupps Co.(Germantown, Ohio), Grupo Tecnal (Sao Paulo, Brazil), Insta Pro (DesMoines, Iowa), French Oil Mill (Piqua, OH), Harburg Freudenberger(previously Krupp Extraktionstechnik) (Hamburg, Germany),Maschinenfabrik Reinartz (Neuss, Germany), Shann Consulting (New SouthWales, Australia) and SKET (Magdeburg, Germany).

Microbial biomass or conditioned feedstock is supplied to the expellerpress via an intake. A rotating feeder screw advances the materialsupplied from the intake into the barrel where it is then compressed byrotation of the worm shaft. Oil extracted from the material is thencollected in an oil pan and then pumped to a storage tank. The remainingspent biomass is then extruded out of the press as a cake and can becollected for additional processing (see subsection G below). The cakemay be pelletized.

The worm shaft is associated with a collar setup and is divided intosections. The worm and collar setup within each section is customizable.The worm shaft is responsible for conveying biomass (feedstock) throughthe press. It may be characterized as having a certain diameter and athread pitch. Changing shaft diameter and pitch can increase or decreasethe pressure and shear stress applied to feedstock as it passes throughthe press. The collar's purpose is to increase the pressure on thefeedstock within the press and also apply a shear stress to the biomass.

The press load in terms of electrical current required to run the pressloaded with microbial biomass (conditioned feedstock) is usually notmore than about 10% of the electrical current required to run the pressempty, and this suggests that the power required to press microbialbiomass (conditioned feedstock disclosed herein is lower than othertypical power requirements from the oil seed industry where the fullpress load is greater than 10% of the electrical current required to runthe press empty of an oil seed feedstock.

The worm shaft preferably is tapered so that its outer diameterincreases along the longitudinal length away from the barrel entrance.This decreases the gap between the worm shaft and the inside of thebarrel thus creating greater pressure and shear stress as the biomasstravels through the barrel. Additionally, the interior of the barrel ismade up of flat steel bars separated by spacers (also referred to asshims), which are set edgewise around the periphery of the barrel, andare held in place by a heavy cradle-type cage. Adjusting the shimbetween the bars controls the gap between the bars which helps theextracted oil to drain as well as also helping to regulate barrelpressure. The shims are often from 0.003″ thick to 0.030″ thick andpreferably from 0.005″ to 0.020″ thick, although other thicknesses mayalso be employed. Additionally, the bars may be adjusted, therebycreating sections within the barrel.

As the feed material is pressed or moved down the barrel, significantheat is generated by friction. In some cases, the amount of heat iscontrolled using a water-jacketed cooling system that surrounds thebarrel. Because of the extreme pressure, oil that is pressed from ascrew press or expeller press contains a proportion of “foots” or solidmaterial from the biomass that flows out with the oil between the bars.The foots can be screened, drained and fed back into the press alongwith unpressed feedstock. Temperature sensors may be disposed at variouslocations around the barrel to monitor and aid in temperature control.Additionally, pressure sensors may also be attached to the barrel atvarious locations to help monitor and control the pressure.

Various operating characteristics of the expeller (screw) press can beexpressed or analyzed as a compression ratio. Compression ratio is theratio of the volume of material displaced per revolution of the wormshaft at the beginning of the barrel divided by the volume of materialdisplaced per revolution of the worm shaft at the end of the barrel. Forexample, due to increasing compression ratios the pressure may be 10 to18 times higher at the end of the barrel as compared with the beginningof the barrel. Internal barrel length may be at least ten times or eventhirteen times the internal barrel diameter. Typical compression ratiofor a screw or expeller press ranges from 1 to 18, depending on the feedmaterial.

Residence time of the feed material in an expeller (screw) press mayaffect the amount of oil recovery. Increased residence time in the pressgives the feedstock more exposure to the shear stress and pressuregenerated by the press, which may yield higher oil recovery. Residencetime of the feedstock depends on the speed at which the press is run andthe length vs. diameter of the screw press (or L/D). The greater theratio of the length of the shaft to the diameter of the shaft, thelonger the residence time of the feedstock (when rotational speed isheld at a constant). In some embodiments, the residence time of thealgal biomass that is being pressed with an expeller press is no morethan 5 to 10 minutes. This residence time for algal biomass is aboutdouble the average residence time for other oil seeds such as soybean,canola or cottonseed.

The resulting pressed solids or cake (spent biomass of reduced oilcontent relative to the feedstock supplied to the screw press) isexpelled from the expeller press through the discharge cone at the endof the barrel/shaft. The choke utilizes a hydraulic system to controlthe exit aperture on the expeller press. A fully optimized oil pressoperation can extract most of the available oil in the oil-bearingmaterial. For example, optimized conditions for oil extraction fromsoybeans using an expeller press leaves about 4-6% residual oil; similaryields can be obtained from microbial biomass (conditioned feedstock) inaccordance with the methods of the invention. A variety of factors canaffect the residual oil content in the pressed cake. These factorsinclude, but are not limited to, the ability of the press to ruptureoil-containing cells and cellular compartments and the composition ofthe oil-bearing material itself, which can have an affinity for theexpelled oil. In some cases, the oil-bearing material may have a highaffinity for the expelled oil and can absorb the expelled oil back intothe material, thereby trapping it. In that event, the oil remaining inthe spent biomass can be re-pressed or subjected to solvent extraction,as described herein, to recover the oil.

It is not necessary to use biological agents to extract oil using anexpeller press, ie: agents such as enzymes that are producedindependently of the microbial biomass. The pressure exerted on theconditioned biomass is the primary mechanism by which oil is releasedfrom oil vesicles in the microbial biomass.

F. Microbial Oil Produced

After the pressing step, the method of the invention results in theextraction of oil and, consequently, the production of extracted oil andspent biomass of reduced oil content relative to the conditionedfeedstock supplied to the pressing step. In various embodiments, thereleased oil contains solid particles of biomass (conditionedfeedstock), and the method further comprises separating the released oilfrom the solid particles.

Contaminants may be present in the oil after pressing (or solventextraction, see subsection G below, or both). In some embodiments, itmay be advantageous to remove these contaminants before subsequent useof the oil (either for food applications or in subsequent chemicalreactions, as in the production of fuels). Fines, or small particulatesfrom the biomass, may be present in the extracted oil. Usually, finesare removed through passing the oil through a filter or some otherprocess that physically separates the particulates from the oil.Optionally, the separated solid particles can be subjected to pressureor solvent extraction to extract any remaining oil therefrom.

Degumming is another process suitable for use in the methods of theinvention that removes contaminants such as phospholipids from the oil.In some embodiments of the invention, degumming of the extracted oil iscombined with refining, bleaching and deodorizing (or RBD). The RBDprocess eliminates or reduces the odor, color and/or taste of theextracted oil. The refining process usually consists of two steps,degumming and a neutralization step that removes the free fatty acids(FFA) in the oil through caustic stripping with sodium hydroxide. Thebleaching step involves mixing the oil with various bleaching clays toabsorb color, trace metals and sulfur compounds. The deodorizing step isa distillation process that occurs at low pressure and high temperature.The oil is put under a vaccum and heated with steam to remove anyleftover taste or odors and FFAs. Deodorizing can also be achieved bytreatment with activated charcoal.

Other methods of removal of contaminants such as heavy metals involvealkai-refining, acid pretreatment and the use of activated clays orzeolites may also be employed in various embodiments of the invention.The oil is mixed at moderate temperatures with small amounts of certainalkaline or ammonia hydroxides and alkaline or ammonia salts in thepresence of a phase transfer catalyst. The Phillips Recycled Oil Program(PROP) technology, developed by the Phillips Petroleum Company, combineschemical demetallisation and hydrogenation to remove contaminants fromoil. The process involves mixing the oil with an aqueous solutionofdiammonium phosphate at an elevated temperature in order to reduce themetal content of the oil. This process leads to chemical reactions thatform metallic phosphates, which can then be removed from the oil byfiltration. Next, the oil is mixed with hydrogen and percolated from abed of clay and passed over an Ni/Mo catalyst in a hydrogenationreactor. This adsorption step removes the remaining traces ofcontaminating compounds, such as sulfur, oxygen, chlorine and nitrogen.

In various embodiments, the extracted oil produced by the methods of theinvention contains no more than 8 ppm chloride, no more than 2 ppmphosphorus, no more than 26 ppm potassium, no more than 12 ppm sodium,and/or no more than 5 ppm sulfur. The oil produced by the process isuseful in a variety of applications, including but not limited to theproduction of fuels such as biodiesel and renewable diesel (see, e.g.,PCT Publication No. 2008/151149 and PCT Application Nos. US09/066141 andUS09/066142, each of which is incorporated herein by reference) and theproduction of food (see, e.g., PCT Application No. US09/060692,incorporated herein by reference).

G. Spent Biomass Produced

The oil extraction methods of the present invention result in theproduction of microbial biomass of reduced oil content (spent biomassalso referred to as pressed cake or pressed biomass) relative to theconditioned feedstock subjected to pressure in the pressing step. Invarious embodiments of the present invention, the oil content in thespent biomass of reduced oil content is at least 45 percent less thanthe oil content of the microbial biomass before the pressing step. Invarious embodiments, the spent biomass of reduced oil content remainingafter the pressing step is pelletized or extruded as a cake. The spentcake, which may be subjected to additional processes, includingadditional conditioning and pressing or solvent-based extraction methodsto extract residual oil in accordance with the invention, is similarlyuseful in a variety of applications, including but not limited to use asfood, particularly for animals, and as a press aid. In variousembodiments of the invention, remaining oil is extracted from the spentbiomass of reduced oil content; in various embodiments, the extractingis performed by subjecting the spent biomass to pressure or byextracting the oil with an organic solvent.

In some instances, the pressed cake contains a range of from less than50% oil to less than 1% oil by weight, including, for example, less than40% oil by weight, less than 20% oil by weight, less than 10%, less than5% oil by weight, and less than 2% oil by weight. In all cases, the oilcontent in the pressed cake is less than the oil content in theunpressed material.

In some embodiments, the spent biomass or pressed cake is collected andrecycled back into the press with fresh conditioned feedstock or drybiomass as a bulking agent pressing aid. In this case, it may benecessary to condition the spent biomass before or after it is admixedwith unpressed feedstock or biomass to make it suitable as a pressingaid. In other embodiments, the spent biomass or pressed cake, which cancontain residual oil and other components, i.e., dietary fiber, issuitable for use as human or animal feed or feed additive. In suchapplications, the spent biomass produced by the methods of the inventionmay be referred to as “meal” or “delipidated meal.”

Thus, spent biomass produced by the methods of the invention is usefulas animal feed for farm animals, e.g., ruminants, poultry, swine, andaquaculture. This delipidated meal, as described above, is microbialbiomass of reduced lipid/oil content, and can be produced through amechanical process (e.g., pressing) or through a solvent extractionprocess (see below), or both. Typically, delipidated meal has less than15% oil by weight. In a preferred embodiment, the delipidated mealgenerated from expeller (screw) pressing of microbial biomass followedby solvent extraction, has an oil content of less than 10% by weight. Asdescribed above, delipidated meal is suitable for use as a bulking agent(press aid). Addtionally, delipidated meal, although of reduced oilcontent, still contains high quality proteins, carbohydrates, fiber,minerals, and other nutrients appropriate for an animal feed. Becausethe cells are predominantly lysed, delipidated meal is easily digested.Delipidated meal can optionally be combined with other ingredients, suchas grain, in an animal feed. Because delipidated meal has a powderyconsistency, it can be pressed into pellets using an extruder orexpanders, which are commercially available.

As noted above, spent biomass, depending on the efficiency of thepressing step, can contain significant amounts of oil. While, in variousembodiments, this oil can be extracted by pressing in accordance withthe methods of the invention (for example, as when the spent biomass isused as a bulking agent), the spent biomass can also be subjected tosolvent extraction to recover more oil from the microbial biomass.

One example of solvent extraction suitable for use in such embodimentsof the invention is hexane solvent extraction. In this embodiment, afterthe oil has been extracted using pressing, the remaining spent biomassis mixed with hexane to extract the remaining oil content. The free oilin the spent microbial biomass forms miscella with the solvent (e.g.,hexane) and is separated from the solids (delipidated biomass meal). Theoil-solvent miscella is filtered and the solvent is evaporated andrecycled for use in future solvent extractions. The delipidated biomassmeal can be desolventized and so rendered suitable for use in animalfeed or feed additive in accordance with the methods of the invention.

Solvent extraction can recover the free oil that is trapped orreabsorbed in the spent microbial biomass; however, solvent extractioncannot recover oil that is still trapped in unbroken/unlysed microbialcells. Microbial biomass that has been conditioned (and lysed) in anextruder or expander but not subjected to the high pressure of a screwpress may also be solvent extracted in order to recover the oil freedfrom the biomass during the conditioning step. Because the efficiency ofsolvent extraction depends on the accessibility of the solvent to thefree oil, increasing the porosity and/or the surface area of thematerial for solvent extraction is important. Ideally, for solventextraction, the spent microbial biomass or pressed cake should contain ahigh percentage of lysed or broken microbial cells, be of porous textureand increased surface area for solvent extraction, should not be highlycompressed or burned, and should not be powdery and dry. In a preferredembodiment, the spent microbial biomass contains at least 85% lysed orbroken microbial cells.

Several types of solvent extractors are used in the art and are suitablefor use with the spent biomass as described above. In one embodiment, acontinuous, percolation solvent extractor is used to extract residualfree oil from the spent microbial biomass. Another method of oilextraction suitable for use in accordance with the methods of theinvention is the supercritical fluid/carbon dioxide extraction method inwhich carbon dioxide is liquefied under pressure and heated to the pointthat it has the properties of both a liquid and a gas. This liquefiedfluid then acts as the solvent in extracting the oil from the spentmicrobial biomass.

Solventless extraction methods known in the art for lipids can also beused for recovering oil from spent biomass in accordance with themethods of the invention. For example, the methods described in U.S.Pat. No. 6,750,048 can be used to recover oil from spent biomassproduced by the methods of the invention. Another suitable solventlessextraction method involves treating the spent biomass with an acid tocreate a liquid slurry. Optionally, the slurry can be sonicated toensure the complete lysis of the microalgae cells in the spent biomass.The lysate produced by acid treatment is preferably created attemperatures above room temperature. Such a lysate, upon centrifugationor settling by gravity, can be separated into layers, one of which is anaqueous:lipid layer. Other layers can include a solid pellet, an aqueouslayer, and a lipid layer. Lipid can be extracted from the emulsion layerby freeze thawing or otherwise cooling the emulsion. In such methods, itis not necessary to add any organic solvent, although in someembodiments it may be advantageous to do so.

The following section describes microorganisms useful for producingoil-containing microbial biomass suitable for use in the methods of theinvention.

III. Microorganisms Useful for Producing Oil and Methods for CulturingThem

The present invention arose in part from the discovery that certainmicroorganisms can be used to produce oil, and hydrocarbon compositionsderived therefrom, economically and in large quantities for use in thetransportation fuel and petrochemical industry, as well as many otherapplications. Suitable microorganisms include microalgae, oleaginousbacteria, oleaginous yeast, and fungi. Acidic transesterfication oflipids yields long-chain fatty acid esters useful as a biodiesel. Otherenzymatic processes can be applied to lipids derived from theseorganisms as described herein to yield fatty acids, aldehydes, alcohols,and alkanes. The present invention also provides methods for cultivatingmicroorganisms such as microalgae to achieve increased productivity oflipids and increased lipid yield.

Microorganisms useful in the invention produce oil (lipids orhydrocarbons) suitable for biodiesel production or as feedstock forindustrial applications. Suitable hydrocarbons for biodiesel productioninclude triacylglycerides (TAGs) containing long-chain fatty acidmolecules. Suitable hydrocarbons for industrial applications, such asmanufacturing, include fatty acids, aldehydes, alcohols, and alkanes. Insome embodiments, suitable fatty acids, or the corresponding primaryalcohols, aldehydes or alkanes, generated by the methods describedherein, contain from at least 8 to at least 35 carbon atoms. Long-chainfatty acids for biodiesel generally contain at least 14 carbon or moreatoms.

Preferred fatty acids, or the corresponding primary alcohols, aldehydes,and alkanes, for industrial applications contain at least 8 or morecarbon atoms. In certain embodiments of the invention, the above fattyacids, as well as the other corresponding hydrocarbon molecules, aresaturated (with no carbon-carbon double or triple bonds);mono-unsaturated (single carbon-carbon double bond); or poly-unsaturated(two or more carbon-carbon double bonds); and are linear (not cyclic);and/or have little or no branching in their structures.

Triacylglycerols containing carbon chain lengths in the C8 to C22 rangecan be produced using the methods of the invention and are preferred fora variety of applications. For surfactants, the preferred TAGs aretypically C10-C14. For biodiesel or renewable diesel, the preferred TAGSare typically C16-C18. For jet fuel, the preferred TAGS are typicallyare C8-C10. For nutrition, the preferred TAGs are C22 polyunsaturatedfatty acids (such as, DHA) and carotenoids (such as astaxanthin).

Any species of organism that produces suitable lipid or hydrocarbon canbe used in the methods of the invention, although microorganisms thatnaturally produce high levels of suitable lipid or hydrocarbon arepreferred. Production of hydrocarbons by microorganisms is reviewed byMetzger et al., Appl Microbiol Biotechnol (2005) 66: 486-496 and A LookBack at the U.S. Department of Energy's Aquatic Species Program:Biodiesel from Algae, NREL/TP-580-24190, John Sheehan, Terri Dunahay,John Benemann and Paul Roessler (1998), incorporated herein byreference.

Considerations affecting the selection of a microorganism for use in theinvention include, in addition to production of suitable hydrocarbon forbiodiesel or for industrial applications: (1) high lipid content as apercentage of cell weight; (2) ease of growth; and (3) ease ofprocessing. In particular embodiments, the microorganism yields cellsthat are at least: about 40%, to 60% or more (including more than 70%)lipid when harvested for oil extraction. For certain applications,organisms that grow heterotrophically (on sugar in the absence of light)or can be engineered to do so, are useful in the methods of theinvention. See U.S. Patent Application Nos. 60/837,839, 61/118,994, SerNos. 11/893,364, and 12/194,389, as well as US Patent ApplicationPublication Nos. 20090004715, 20090047721, 20090011480, 20090035842,20090061493, and 20090148918; PCT Application Nos. 2009/066141 and2009/066142; and PCT Publication No.2008/151149, each of which isincorporated herein by reference in their entireties. For applicationsin which an organism will be genetically modified, the ease oftransformation and availability of selectable markers and promoters,constitutive and/or inducible, that are functional in the microorganismwill affect the selection of the organism to be modified.

Naturally occurring microalgae are preferred microorganisms for use inthe methods of the invention. Thus, in various preferred embodiments ofthe present invention, the microorganism producing a lipid—themicroorganism from which oil is extracted, recovered, or obtained—is amicroalgae. Examples of genera and species of microalgae that can beused in the methods of the present invention include, but are notlimited to, the following genera and species microalgae.

TABLE 1 Microalgae. Achnanthes orientalis, Agmenellum, Amphiprorahyaline, Amphora coffeiformis, Amphora coffeiformis linea, Amphoracoffeiformis punctata, Amphora coffeiformis taylori, Amphoracoffeiformis tenuis, Amphora delicatissima, Amphora delicatissimacapitata, Amphora sp., Anabaena, Ankistrodesmus, Ankistrodesmusfalcatus, Boekelovia hooglandii, Borodinella sp., Botryococcus braunii,Botryococcus sudeticus, Bracteoccocus aerius, Bracteococcus sp.,Bracteacoccus grandis, Bracteacoccus cinnabarinas, Bracteococcus minor,Bracteococcus medionucleatus, Carteria, Chaetoceros gracilis,Chaetoceros muelleri, Chaetoceros muelleri subsalsum, Chaetoceros sp.,Chlorella anitrata, Chlorella Antarctica, Chlorella aureoviridis,Chlorella candida, Chlorella capsulate, Chlorella desiccate, Chlorellaellipsoidea, Chlorella emersonii, Chlorella fusca, Chlorella fusca var.vacuolata, Chlorella glucotropha, Chlorella infusionum, Chlorellainfusionum var. actophila, Chlorella infusionum var. auxenophila,Chlorella kessleri, Chlorella lobophora (strain SAG 37.88), Chlorellaluteoviridis, Chlorella luteoviridis var. aureoviridis, Chlorellaluteoviridis var. lutescens, Chlorella miniata, Chlorella cf.minutissima, Chlorella minutissima, Chlorella mutabilis, Chlorellanocturna, Chlorella ovalis, Chlorella parva, Chlorella photophila,Chlorella pringsheimii, Chlorella protothecoides (including any of UTEXstrains 1806, 411, 264, 256, 255, 250, 249, 31, 29, 25), Chlorellaprotothecoides var. acidicola, Chlorella regularis, Chlorella regularisvar. minima, Chlorella regularis var. umbricata, Chlorella reisiglii,Chlorella saccharophila, Chlorella saccharophila var. ellipsoidea,Chlorella salina, Chlorella simplex, Chlorella sorokiniana, Chlorellasp., Chlorella sphaerica, Chlorella stigmatophora, Chlorella vanniellii,Chlorella vulgaris, Chlorella vulgaris f. tertia, Chlorella vulgarisvar. autotrophica, Chlorella vulgaris var. viridis, Chlorella vulgarisvar. vulgaris, Chlorella vulgaris var. vulgaris f. tertia, Chlorellavulgaris var. vulgaris f. viridis, Chlorella xanthella, Chlorellazofingiensis, Chlorella trebouxioides, Chlorella vulgaris, Chlorococcuminfusionum, Chlorococcum sp., Chlorogonium, Chroomonas sp.,Chrysosphaera sp., Cricosphaera sp., Crypthecodinium cohnii, Cryptomonassp., Cyclotella cryptica, Cyclotella meneghiniana, Cyclotella sp.,Dunaliella sp., Dunaliella bardawil, Dunaliella bioculata, Dunaliellagranulate, Dunaliella maritime, Dunaliella minuta, Dunaliella parva,Dunaliella peircei, Dunaliella primolecta, Dunaliella salina, Dunaliellaterricola, Dunaliella tertiolecta, Dunaliella viridis, Dunaliellatertiolecta, Eremosphaera viridis, Eremosphaera sp., Ellipsoidon sp.,Euglena, Franceia sp., Fragilaria crotonensis, Fragilaria sp., Gleocapsasp., Gloeothamnion sp., Hymenomonas sp., Isochrysis aff. galbana,Isochrysis galbana, Lepocinclis, Micractinium, Micractinium (UTEX LB2614), Monoraphidium minutum, Monoraphidium sp., Nannochloris sp.,Nannochloropsis salina, Nannochloropsis sp., Navicula acceptata,Navicula biskanterae, Navicula pseudotenelloides, Navicula pelliculosa,Navicula saprophila, Navicula sp., Neochloris oleabundans, Nephrochlorissp., Nephroselmis sp., Nitschia communis, Nitzschia alexandrina,Nitzschia communis, Nitzschia dissipata, Nitzschia frustulum, Nitzschiahantzschiana, Nitzschia inconspicua, Nitzschia intermedia, Nitzschiamicrocephala, Nitzschia pusilla, Nitzschia pusilla elliptica, Nitzschiapusilla monoensis, Nitzschia quadrangular, Nitzschia sp., Ochromonassp., Oocystis parva, Oocystis pusilla, Oocystis sp., Oscillatorialimnetica, Oscillatoria sp., Oscillatoria subbrevis, Parachlorellabeijerinckii, Parachlorella kessleri, Pascheria acidophila, Pavlova sp.,Phagus, Phormidium, Platymonas sp., Pleurochrysis carterae,Pleurochrysis dentate, Pleurochrysis sp., Prototheca stagnora,Prototheca portoricensis, Prototheca moriformis, Prototheca wickerhamii,Prototheca zopfii, Pseudochlorella aquatica, Pyramimonas sp.,Pyrobotrys, Sarcinoid chrysophyte, Scenedesmus armatus, Scenedesmusrubescens, Schizochytrium, Spirogyra, Spirulina platensis, Stichococcussp., Synechococcus sp., Tetraedron, Tetraselmis sp., Tetraselmissuecica, Thalassiosira weissflogii, and Viridiella fridericiana.

In various preferred embodiments of the present invention, themicroorganism producing a lipid or a microorganism from which oil can beextracted, recovered, or obtained is an organism of a species of thegenus Chlorella. In various preferred embodiments, the microalgae isChlorella protothecoides, Chlorella ellipsoidea, Chlorella minutissima,Chlorella zofinienesi, Chlorella luteoviridis, Chlorella kessleri,Chlorella sorokiniana, Chlorella fusca var. vacuolate Chlorella sp.,Chlorella cf. minutissima or Chlorella emersonii. Chlorella is a genusof single-celled green algae, belonging to the phylum Chlorophyta. It isspherical in shape, about 2 to 10 μm in diameter, and is withoutflagella. Some species of Chlorella are naturally heterotrophic.Chlorella, particularly Chlorella protothecoides, is a preferredmicroorganism for use in the invention because of its high compositionof lipid and its ability to grow heterotrophically.

Chlorella, preferably, Chlorella protothecoides, Chlorella minutissima,or Chlorella emersonii, can be genetically engineered to express one ormore heterologous genes (“transgenes”). Examples of expression oftransgenes in, e.g., Chlorella, can be found in the literature (see forexample Current Microbiology Vol. 35 (1997), pp. 356-362; Sheng Wu GongCheng Xue Bao. 2000 July;16(4):443-6; Current Microbiology Vol. 38(1999), pp. 335-341; Appl Microbiol Biotechnol (2006) 72: 197-205;Marine Biotechnology 4, 63-73, 2002; Current Genetics 39:5, 365-370(2001); Plant Cell Reports 18:9, 778-780, (1999); Biologia Plantarium42(2): 209-216, (1999); Plant Pathol. J 21(1): 13-20, (2005)), and suchreferences are incorporated herein by reference as teaching variousmethods and materials for introducing and expressing genes of interestin such organisms, as the patent applications referenced above. Otherlipid-producing microalgae can be engineered as well, includingprokaryotic Microalgae (see Kalscheuer et al., Applied Microbiology andBiotechnology, Volume 52, Number 4/October, 1999), which are suitablefor use in the methods of the invention.

Species of Chlorella suitable for use in the invention can also beidentified by a method that involves amplification of certain targetregions of the genome. For example, identification of a specificChlorella species or strain can be achieved through amplification andsequencing of nuclear and/or chloroplast DNA using primers andmethodology using any region of the genome, such as, for example, themethods described in Wu et al., Bot. Bull. Acad. Sin. 42:115-121 (2001).Identification of Chlorella spp. isolates using ribosomal DNA sequences.Well established methods of phylogenetic analysis, such as amplificationand sequencing of ribosomal internal transcribed spacer (ITS1 and ITS2rDNA), 18S rRNA, and other conserved genomic regions can be used bythose skilled in the art to identify species of not only Chlorella, butother oil and lipid producing organisms capable of using the methodsdisclosed herein. For examples of methods of identification andclassification of algae see Genetics, 170(4):1601-10 (2005) and RNA,11(4):361-4 (2005).

Genomic DNA comparison can also be used to identify suitable species ofmicroalgae for use in the methods of the present invention. Regions ofconserved DNA, including but not limited to DNA encoding 23S rRNA, canbe amplified from microalgal species and compared to consensus sequencesto screen for microalgal species that are taxonomically related to apreferred microalgae for use in the methods of the present invention.Similar genomic DNA comparisons can also be used to identify suitablespecies of oleaginous yeast for use in the methods of the presentinvention. Regions of conserved genomic DNA, such as, but not limited toconserved genomic sequences between three prime regions of fungal 18Sand five prime regions of fungal 26S rRNA genes can be amplified fromoleaginous yeast species that may be, for example, taxonomically relatedto the preferred oleaginous yeast species used in the present inventionand compared to the corresponding regions of those preferred species.Example 13 describes genomic sequencing of conserved 3′ regions offungal 18S and 5′regions of fungal 26S rRNA for 48 strains of oleaginousyeasts and the genomic sequences are listed as SEQ ID NOs: 37-69.

In some embodiments, oleaginous yeast preferred for use in the methodsof the present invention have genomic DNA sequences encoding for fungal18S and 26S rRNA genomic sequence with at least 75%, 85% or 95%nucleotide identity to one or more of SEQ ID NOs: 37-69.

In some embodiments, microalgae preferred for use in the methods of thepresent invention have genomic DNA sequences encoding 23S rRNA that areat least 99%, or at least 95%, or at least 90%, or at least 85%identical to a 23S rRNA sequences of a Chlorella species.

Prototheca is a genus of single-cell microalgae believed to be anon-photosynthetic mutant of Chlorella. While Chlorella can obtain itsenergy through photosynthesis, species of the genus Prototheca areobligate heterotrophs. Prototheca are spherical in shape, about 2 to 15micrometers in diameter, and lack flagella. In various preferredembodiments, the microalgae used in the methods of the invention isselected from the following species of Prototheca: Prototheca stagnora,Prototheca portoricensis, Prototheca moriformis, Prototheca wickerhamiiand Prototheca zopfii.

In some embodiments, microalgae preferred for use in the methods of thepresent invention have genomic DNA sequences encoding 23S rRNA that haveat least 99%, or at least 95%, or at least 90%, or at least 85%identical to a 23S rRNA sequence of a Prototheca species.

In addition to Prototheca and Chlorella, other microalgae can be used inaccordance with the methods of the present invention. In variouspreferred embodiments, the microalgae is selected from a genus orspecies from any of the following genera and species:

-   Parachlorella kessleri, Parachlorella beijerinckii, Neochloris    oleabundans, Bracteacoccus grandis, Bracteacoccus cinnabarinas,    Bracteococcus aerius, Bracteococcus sp. or Scenedesmus rebescens.    Other non-limiting examples of microalgae (including Chlorella) are    listed in Table 1, above.

In addition to microalgae, oleaginous yeast can accumulate more than 20%of their dry cell weight as lipid and so are useful in the methods ofthe invention. In one preferred embodiment of the present invention, amicroorganism producing a lipid or a microorganism from which oil can beextracted, recovered, or obtained, is an oleaginous yeast. Examples ofoleaginous yeast that can be used in the methods of the presentinvention include, but are not limited to, the oleaginous yeast listedin Table 2. Illustrative methods for the cultivation of oleaginous yeast(Yarrowia lipolytica and Rhodotorula graminis) in order to achieve highoil content are provided in the examples below.

TABLE 2 Oleaginous Yeast. Candida apicola, Candida sp., Cryptococcuscurvatus, Cryptococcus terricolus, Debaromyces hansenii, Endomycopsisvernalis, Geotrichum carabidarum, Geotrichum cucujoidarum, Geotrichumhisteridarum, Geotrichum silvicola, Geotrichum vulgare, Hyphopichiaburtonii, Lipomyces lipofer, Lypomyces orentalis, Lipomyces starkeyi,Lipomyces tetrasporous, Pichia mexicana, Rodosporidium sphaerocarpum,Rhodosporidium toruloides Rhodotorula aurantiaca, Rhodotoruladairenensis, Rhodotorula diffluens, Rhodotorula glutinus, Rhodotorulaglutinis var. glutinis, Rhodotorula gracilis, Rhodotorula graminisRhodotorula minuta, Rhodotorula mucilaginosa, Rhodotorula mucilaginosavar. mucilaginosa, Rhodotorula terpenoidalis, Rhodotorula toruloides,Sporobolomyces alborubescens, Starmerella bombicola, Torulasporadelbruekii, Torulaspora pretoriensis, Trichosporon behrend, Trichosporonbrassicae, Trichosporon domesticum, Trichosporon laibachii, Trichosporonloubieri, Trichosporon loubieri var. loubieri, Trichosporonmontevideense, Trichosporon pullulans, Trichosporon sp., WickerhamomycesCanadensis, Yarrowia lipolytica, and Zygoascus meyerae.

In one preferred embodiment of the present invention, a microorganismproducing a lipid or a microorganism from which a lipid can beextracted, recovered or obtained, is a fungus. Examples of fungi thatcan be used in the methods of the present invention include, but are notlimited to, the fungi listed in Table 3.

TABLE 3 Oleaginous Fungi. Mortierella, Mortierrla vinacea, Mortierellaalpine, Pythium debaryanum, Mucor circinelloides, Aspergillus ochraceus,Aspergillus terreus, Pennicillium iilacinum, Hensenulo, Chaetomium,Cladosporium, Malbranchea, Rhizopus, and Pythium

Thus, in one embodiment of the present invention, the microorganism usedfor the production of microbial biomass for use in the methods of theinvention is a fungus. Examples of suitable fungi (e.g., Mortierellaalpine, Mucor circinelloides, and Aspergillus ochraceus) include thosethat have been shown to be amenable to genetic manipulation, asdescribed in the literature (see, for example, Microbiology, July;153(Pt.7): 2013-25 (2007); Mol Genet Genomics, June; 271(5): 595-602(2004); Curr Genet, March;21(3):215-23 (1992); Current Microbiology,30(2):83-86 (1995); Sakuradani, NISR Research Grant, “Studies ofMetabolic Engineering of Useful Lipid-producing Microorganisms” (2004);and PCT/JP2004/012021).

In other embodiments of the present invention, a microorganism producinga lipid or a microorganism from which oil can be extracted, recovered,or obtained is an oleaginous bacterium. Oleaginous bacteria are bacteriathat can accumulate more than 20% of their dry cell weight as lipid.Species of oleaginous bacteria for use in the methods of the presentinvention, include species of the genus Rhodococcus, such as Rhodococcusopacus and Rhodococcus sp. Methods of cultivating oleaginous bacteria,such as Rhodococcus opacus, are known in the art (see Waltermann, etal., (2000) Microbiology, 146: 1143-1149). Illustrative methods forcultivating Rhodococcus opacus to achieve high oil content are providedin the examples below.

To produce oil-containing microbial biomass suitable for use in themethods of the invention, microorganisms are cultured for production ofoil (e.g., hydrocarbons, lipids, fatty acids, aldehydes, alcohols andalkanes). This type of culture is typically first conducted on a smallscale and, initially, at least, under conditions in which the startingmicroorganism can grow. For example, if the starting microorganism is aphotoautotroph, the initial culture is conducted in the presence oflight. The culture conditions can be changed if the microorganism isevolved or engineered to grow independently of light. Culture forpurposes of hydrocarbon production is preferentially conducted on alarge scale. Preferably, a fixed carbon source is present in excess. Theculture can also be exposed to light some or all of the time, if desiredor beneficial.

Microalgae can be cultured in liquid media. The culture can be containedwithin a bioreactor. Optionally, the bioreactor does not allow light toenter. Alternatively, microalgae can be cultured in photobioreactorsthat contain a fixed carbon source and allow light to strike the cells.For microalgae cells that can utilize light as an energy source,exposure of those cells to light, even in the presence of a fixed carbonsource that the cells transport and utilize (i.e., mixotrophic growth),nonetheless accelerates growth compared to culturing those cells in thedark. Culture condition parameters can be manipulated to optimize totaloil production, the combination of hydrocarbon species produced, and/orproduction of a particular hydrocarbon species. In some instances, it ispreferable to culture cells in the dark, such as, for example, whenusing extremely large (40,000 liter and higher) fermentors that do notallow light to strike a significant proportion (or any) of the culture.

Microalgal culture medium typically contains components such as a fixednitrogen source, trace elements, optionally a buffer for pH maintenance,and phosphate. Components in addition to a fixed carbon source, such asacetate or glucose, may include salts such as sodium chloride,particularly for seawater microalgae. Examples of trace elements includezinc, boron, cobalt, copper, manganese, and molybdenum, in, for example,the respective forms of ZnCl₂, H₃BO₃, CoCl₂.6H₂O, CuCl₂.2H₂O, MnCl₂.4H₂Oand (NH₄)₆Mo₇O₂₄.4H₂O. Other culture parameters can also be manipulated,such as the pH of the culture media, the identity and concentration oftrace elements and other media constituents.

For organisms able to grow on a fixed carbon source, the fixed carbonsource can be, for example, glucose, fructose, sucrose, galactose,xylose, mannose, rhamnose, N-acetylglucosamine, glycerol, floridoside,glucuronic acid, and/or acetate. The one or more exogenously providedfixed carbon source(s) can be supplied to the culture medium at aconcentration of from at least about 50 μM to at least 500 mM, and atvarious amounts in that range (i.e., 100 μM, 500 μM, 5 mM, 50 mM).

Certain microalgae can be grown in the presence of light. The number ofphotons striking a culture of such microalgae cells can be manipulated,as well as other parameters such as the wavelength spectrum and ratio ofdark:light hours per day. Microalgae can also be cultured in naturallight, as well as simultaneous and/or alternating combinations ofnatural light and artificial light. For example, microalgae of the genusChlorella can be cultured under natural light during daylight hours andunder artificial light during night hours.

The gas content of a photobioreactor to grow microorganisms likemicroalgae can be manipulated. Part of the volume of a photobioreactorcan contain gas rather than liquid. Gas inlets can be used to pump gasesinto the photobioreactor. Any gas can be pumped into a photobioreactor,including air, air/CO₂ mixtures, noble gases such as argon and others.The rate of entry of gas into a photobioreactor can also be manipulated.Increasing gas flow into a photobioreactor increases the turbidity of aculture of microalgae. Placement of ports conveying gases into aphotobioreactor can also affect the turbidity of a culture at a givengas flow rate. Air/CO₂ mixtures can be modulated to generate optimalamounts of CO₂ for maximal growth by a particular organism. Microalgaegrow significantly faster in the light under, for example, 3% CO₂/97%air than in 100% air. 3% CO₂/97% air has approximately 100-fold more CO₂than found in air. For example, air:CO₂ mixtures in a range of fromabout 99.75% air:0.25% CO₂ to 95.00% air:5.0% CO₂ can be infused into abioreactor or photobioreactor.

Microalgae cultures can also be subjected to mixing using devices suchas spinning blades and impellers, rocking of a culture, stir bars,infusion of pressurized gas, and other instruments; such methods can beused to ensure that all cells in a photobioreactor are exposed to lightbut of course find application with cultures of cells that are not usinglight as an energy source.

Some microalgae species can grow by utilizing a fixed carbon source,such as glucose or acetate, in the absence of light. Such growth isknown as heterotrophic growth. For Chlorella protothecoides, forexample, heterotrophic growth results in high production of biomass andaccumulation of high lipid content. Thus, an alternative tophotosynthetic growth and propagation of microorganisms, as describedabove, is the use of heterotrophic growth and propagation ofmicroorganisms, under conditions in which a fixed carbon source providesenergy for growth and lipid accumulation. In some embodiments, the fixedcarbon energy source comprises cellulosic material, includingdepolymerized cellulosic material, a 5-carbon sugar, or a 6-carbonsugar.

Methods for the growth and propagation of Chlorella protothecoides tohigh oil levels as a percentage of dry weight have been reported (seefor example Miao and Wu, J. Biotechnology, 2004, 11:85-93 and Miao andWu, Biosource Technology (2006) 97:841-846, reporting methods forobtaining 55% oil dry cell weight).

PCT Publication W02008/151149, incorporated herein by reference,describes preferred growth conditions for Chlorella. Multiple species ofChlorella and multiple strains within a species can be grown in thepresence of glycerol. The aforementioned patent application describesculture parameters incorporating the use of glycerol for fermentation ofmultiple genera of microalgae. Multiple Chlorella species and strainsproliferate very well on not only purified reagent-grade glycerol, butalso on acidulated and non-acidulated glycerol byproduct from biodieseltransesterification. In some instances, microalgae, such as Chlorellastrains, undergo cell division faster in the presence of glycerol thanin the presence of glucose. In these instances, two-stage growthprocesses in which cells are first fed glycerol to increase celldensity, and are then fed glucose to accumulate lipids can improve theefficiency with which lipids are produced.

Other feedstocks for culturing microalgae under heterotrophic growthconditions for purposes of the present invention include mixtures ofglycerol and glucose, mixtures of glucose and xylose, mixtures offructose and glucose, sucrose, glucose, fructose, xylose, arabinose,mannose, galactose, acetate, and molasses. Other suitable feedstocksinclude corn stover, sugar beet pulp, and switchgrass in combinationwith depolymerization enzymes.

For lipid and oil production, cells, including recombinant cells, aretypically fermented in large quantities. The culturing may be in largeliquid volumes, such as in suspension cultures as an example. Otherexamples include starting with a small culture of cells which expandinto a large biomass in combination with cell growth and propagation aswell as lipid (oil) production. Bioreactors or steel fermentors can beused to accommodate large culture volumes. For these fermentations, useof photosynthetic growth conditions may be impossible or at leastimpractical and inefficient, so heterotrophic growth conditions may bepreferred.

Appropriate nutrient sources for culture in a fermentor forheterotrophic growth conditions include raw materials such as one ormore of the following: a fixed carbon source such as glucose, cornstarch, depolymerized cellulosic material, sucrose, sugar cane, sugarbeet, lactose, milk whey, molasses, or the like; a nitrogen source, suchas protein, soybean meal, cornsteep liquor, ammonia (pure or in saltform), nitrate or nitrate salt; and a phosphorus source, such asphosphate salts. Additionally, a fermentor for heterotrophic growthconditions allows for the control of culture conditions such astemperature, pH, oxygen tension, and carbon dioxide levels. Optionally,gaseous components, like oxygen or nitrogen, can be bubbled through aliquid culture. Other starch (glucose) sources include wheat, potato,rice, and sorghum. Other carbon sources include process streams such astechnical grade glycerol, black liquor, and organic acids such asacetate, and molasses. Carbon sources can also be provided as a mixture,such as a mixture of sucrose and depolymerized sugar beet pulp.

A fermentor for heterotrophic growth conditions can be used to allowcells to undergo the various phases of their physiological cycle. As anexample, an inoculum of lipid-producing cells can be introduced into amedium followed by a lag period (lag phase) before the cells begin topropagate. Following the lag period, the propagation rate increasessteadily and enters the log, or exponential, phase. The exponentialphase is in turn followed by a slowing of propagation due to decreasesin nutrients such as nitrogen, increases in toxic substances, and quorumsensing mechanisms. After this slowing, propagation stops, and the cellsenter a stationary phase or steady growth state, depending on theparticular environment provided to the cells.

In one heterotrophic culture method useful for purposes of the presentinvention, microorganisms are cultured using depolymerized cellulosicbiomass as a feedstock. As opposed to other feedstocks that can be usedto culture microorganisms, such as corn starch or sucrose from sugarcane or sugar beets, cellulosic biomass (depolymerized or otherwise) isnot suitable for human consumption. Cellulosic biomass (e.g., stover,such as corn stover) is inexpensive and readily available; however,attempts to use this material as a feedstock for yeast have failed. Inparticular, such feedstocks have been found to be inhibitory to yeastgrowth, and yeast cannot use the 5-carbon sugars produced fromcellulosic materials (e.g., xylose from hemi-cellulose). By contrast,microalgae can proliferate on depolymerized cellulosic material.Accordingly, the invention contemplates methods of culturing amicroalgae under heterotrophic growth conditions in the presence of acellulosic material and/or a 5-carbon sugar. Cellulosic materialsgenerally include: 40-60% cellulose; 20-40% hemicellulose; and 10-30%lignin.

Suitable cellulosic materials include residues from herbaceous and woodyenergy crops, as well as agricultural crops, i.e., the plant parts,primarily stalks and leaves typically not removed from the fields withthe primary food or fiber product. Examples include agricultural wastessuch as sugarcane bagasse, rice hulls, corn fiber (including stalks,leaves, husks, and cobs), wheat straw, rice straw, sugar beet pulp,citrus pulp, citrus peels; forestry wastes such as hardwood and softwoodthinnings, and hardwood and softwood residues from timber operations;wood wastes such as saw mill wastes (wood chips, sawdust) and pulp millwaste; urban wastes such as paper fractions of municipal solid waste,urban wood waste and urban green waste such as municipal grassclippings; and wood construction waste. Additional cellulosics includededicated cellulosic crops such as switchgrass, hybrid poplar wood, andmiscanthus, fiber cane, and fiber sorghum. Five-carbon sugars that areproduced from such materials include xylose.

Some microbes are able to process cellulosic material and directlyutilize cellulosic materials as a carbon source. However, cellulosicmaterial may need to be treated to increase the accessible surface areaor for the cellulose to be first broken down as a preparation formicrobial utilization as a carbon source. Ways of preparing orpretreating cellulosic material for enzyme digestion are well known inthe art. The methods are divided into two main categories: (1) breakingapart the cellulosic material into smaller particles to increase theaccessible surface area; and (2) chemically treating the cellulosicmaterial to create a useable substrate for enzyme digestion.

Methods for increasing the accessible surface area include steamexplosion, which involves the use of steam at high temperatures to breakapart cellulosic materials. Because of the high temperature requirementof this process, some of the sugars in the cellulosic material may belost, thus reducing the available carbon source for enzyme digestion(see for example, Chahal, D. S. et al., Proceedings of the 2^(nd) WorldCongress of Chemical Engineering; (1981) and Kaar et al., Biomass andBioenergy (1998) 14(3): 277-87) Ammonia explosion allows for explosionof cellulosic material at a lower temperature, but is more costly toperform and the ammonia might interfere with subsequent enzyme digestionprocesses (see for example, Dale, B. E. et al., Biotechnology andBioengineering (1982); 12: 31-43). Another explosion technique involvesthe use of supercritical carbon dioxide explosion to break thecellulosic material into smaller fragments (see for example, Zheng etal., Biotechnology Letters (1995); 17(8): 845-850).

Methods for chemically treating the cellulosic material to createuseable substrates for enzyme digestion are also known in the art. U.S.Pat. No. 7,413,882, incorporated herein by reference, describes the useof genetically engineered microbes that secrete beta-glucosidase intothe fermentation broth and treating cellulosic material with thefermentation broth to enhance the hydrolysis of cellulosic material intoglucose. Cellulosic material can also be treated with strong acids andbases to aid subsequent enzyme digestion. U.S. Pat. No. 3,617,431,incorporated herein by reference, describes the use of alkalinedigestion to breakdown cellulosic materials.

Microorganisms can possess both the ability to utilize an otherwiseinedible feedstock, such as cellulosic material or glycerol, as a carbonsource (or a pre-treated cellulosic material as a carbon source) and thenatural ability to produce edible oils. By utilizing both of theseproperties, cellulosic material or glycerol, which is normally not partof the human food chain (as opposed to corn glucose and sucrose fromsugar cane and sugar beet, which are food compositions suitable forhuman consumption) can be converted into high nutrition, edible oils,which can provide nutrients and calories as part of the daily human (oranimal) diet. In this manner, previously inedible feedstock can beturned into high nutrition edible oils and other food products and foodcompositions that contain these high nutrition edible oils, as well asoils useful for other purposes.

Bioreactors can be employed for heterotrophic growth and propagationmethods. As will be appreciated, provisions made to make light availableto the cells in photosynthetic growth methods are unnecessary when usinga fixed-carbon source in the heterotrophic growth and propagationmethods described herein.

The specific examples of process conditions and heterotrophic growth andpropagation methods described herein can be combined in any suitablemanner to improve efficiencies of microbial growth and lipid production.For example, microbes having a greater ability to utilize any of theabove-described feedstocks for increased proliferation and/or lipidproduction may be used in the methods of the invention.

Mixotrophic growth involves the use of both light and fixed carbonsource(s) as energy sources for cultivating cells. Mixotrophic growthcan be conducted in a photobioreactor. Microalgae can be grown andmaintained in closed photobioreactors made of different types oftransparent or semitransparent material. Such material can includePlexiglass® enclosures, glass enclosures, bags made from substances suchas polyethylene, transparent or semi-transparent pipes and othermaterial. Microalgae can be grown and maintained in openphotobioreactors such as raceway ponds, settling ponds and othernon-enclosed containers. The following discussion of photobioreactorsuseful for mixotrophic growth conditions is applicable to photosyntheticgrowth conditions as well.

Photobioreactors can have ports allowing entry of gases, solids,semisolids, and liquids into the chamber containing the microalgae.Ports are usually attached to tubing or other means of conveyingsubstances. Gas ports, for example, convey gases into the culture.Pumping gases into a photobioreactor can serve both to feed cells CO₂and other gases and to aerate the culture and therefore generateturbidity. The amount of turbidity of a culture varies as the number andposition of gas ports is altered. For example, gas ports can be placedalong the bottom of a cylindrical polyethylene bag. Microalgae growfaster when CO₂ is added to air and bubbled into a photobioreactor. Forexample, a 5% CO₂:95% air mixture can be infused into a photobioreactorcontaining Botryococcus cells for such purposes (see for example J AgricFood Chem. 54(13):4593-9 (2006); J Biosci Bioeng. 87(6):811-5 (1999);and J Nat Prod. 66(6):772-8 (2003)).

Photobioreactors can be exposed to one or more light sources to providemicroalgae with light as an energy source via light directed to asurface of the photobioreactor. Preferably the light source provides anintensity that is sufficient for the cells to grow, but not so intenseas to cause oxidative damage or cause a photoinhibitive response. Insome instances a light source has a wavelength range that mimics orapproximately mimics the range of the sun. In other instances adifferent wavelength range is used. Photobioreactors can be placedoutdoors or in a greenhouse or other facility that allows sunlight tostrike the surface. Preferred photon intensities for species of thegenus Botryococcus are between 25 and 500 μE m⁻² s⁻¹ (see for examplePhotosynth Res. 84(1-3):21-7 (2005)).

As noted above, photobioreactors preferably have one or more ports thatallow media entry. It is not necessary that only one substance enter orleave a port. For example, a port can be used to flow culture media intothe photobioreactor and then later can be used for sampling, gas entry,gas exit, or other purposes. In some instances, a photobioreactor isfilled with culture media at the beginning of a culture, and no moregrowth media is infused after the culture is inoculated. In other words,the microalgal biomass is cultured in an aqueous medium for a period oftime during which the microalgae reproduce and increase in number;however, quantities of aqueous culture medium are not flowed through thephotobioreactor throughout the time period. Thus in some embodiments,aqueous culture medium is not flowed through the photobioreactor afterinoculation.

In other instances, culture media can be flowed though thephotobioreactor throughout the time period during which the microalgaereproduce and increase in number. In some embodiments media is infusedinto the photobioreactor after inoculation but before the cells reach adesired density. In other words, a turbulent flow regime of gas entryand media entry is not maintained for reproduction of microalgae until adesired increase in number of said microalgae has been achieved.

Photobioreactors typically have one or more ports that allow gas entry.Gas can serve to both provide nutrients such as CO₂ as well as toprovide turbulence in the culture media. Turbulence can be achieved byplacing a gas entry port below the level of the aqueous culture media sothat gas entering the photobioreactor bubbles to the surface of theculture. One or more gas exit ports allow gas to escape, therebypreventing pressure buildup in the photobioreactor. Preferably a gasexit port leads to a “one-way” valve that prevents contaminatingmicroorganisms from entering the photobioreactor. In some instances,cells are cultured in a photobioreactor for a period of time duringwhich the microalgae reproduce and increase in number, however aturbulent flow regime with turbulent eddies predominantly throughout theculture media caused by gas entry is not maintained for all of theperiod of time. In other instances a turbulent flow regime withturbulent eddies predominantly throughout the culture media caused bygas entry can be maintained for all of the period of time during whichthe microalgae reproduce and increase in number. In some instances apredetermined range of ratios between the scale of the photobioreactorand the scale of eddies is not maintained for the period of time duringwhich the microalgae reproduce and increase in number. In otherinstances such a range can be maintained.

Photobioreactors typically have at least one port that can be used forsampling the culture. Preferably, a sampling port can be used repeatedlywithout altering compromising the axenic nature of the culture. Asampling port can be configured with a valve or other device that allowsthe flow of sample to be stopped and started. Alternatively a samplingport can allow continuous sampling. Photobioreactors also typically haveat least one port that allows inoculation of a culture. Such a port canalso be used for other purposes such as media or gas entry.

Microorganisms useful in accordance with the methods of the presentinvention are found in various locations and environments throughout theworld. As a consequence of their isolation from other species and theirresulting evolutionary divergence, the particular growth medium foroptimal growth and generation of oil and/or lipid from any particularspecies of microbe may need to be experimentally determined. In somecases, certain strains of microorganisms may be unable to grow on aparticular growth medium because of the presence of some inhibitorycomponent or the absence of some essential nutritional requirementrequired by the particular strain of microorganism. There are a varietyof methods known in the art for culturing a wide variety of species ofmicroalgae to accumulate high levels of lipid as a percentage of drycell weight, and methods for determining optimal growth conditions forany species of interest are also known in the art.

Solid and liquid growth media are generally available from a widevariety of sources, and instructions for the preparation of particularmedia that is suitable for a wide variety of strains of microorganismscan be found, for example, online at http://www.utex.org/, a sitemaintained by the University of Texas at Austin for its culturecollection of algae (UTEX). For example, various fresh water and saltwater media include those shown in Table 4.

TABLE 4 Algal Media. Fresh Water Media Salt Water Media ½ CHEV DiatomMedium 1% F/2 ⅓ CHEV Diatom Medium ½ Enriched Seawater Medium ⅕ CHEVDiatom Medium ½ Erdschreiber Medium 1:1 DYIII/PEA + Gr+ ½ Soil +Seawater Medium ⅔ CHEV Diatom Medium ⅓ Soil + Seawater Medium 2X CHEVDiatom Medium ¼ ERD Ag Diatom Medium ¼ Soil + Seawater Medium AllenMedium ⅕ Soil + Seawater Medium BG11-1 Medium ⅔ Enriched Seawater MediumBold 1NV Medium 20% Allen + 80% ERD Bold 3N Medium 2X Erdschreiber'sMedium Botryococcus Medium 2X Soil + Seawater Medium Bristol Medium 5%F/2 Medium CHEV Diatom Medium 5/3 Soil + Seawater Agar Medium Chu'sMedium Artificial Seawater Medium CR1 Diatom Medium BG11-1 + .36% NaClMedium CR1+ Diatom Medium BG11-1 + 1% NaCl Medium CR1-S Diatom MediumBold 1NV:Erdshreiber (1:1) Cyanidium Medium Bold 1NV:Erdshreiber (4:1)Cyanophycean Medium Bristol-NaCl Medium Desmid Medium DasycladalesSeawater Medium DYIII Medium Enriched Seawater Medium Euglena MediumErdschreiber's Medium HEPES Medium ES/10 Enriched Seawater Medium JMedium ES/2 Enriched Seawater Medium Malt Medium ES/4 Enriched SeawaterMedium MES Medium F/2 Medium Modified Bold 3N Medium F/2 + NH4 ModifiedCOMBO Medium LDM Medium N/20 Medium Modified 2 X CHEV Ochromonas MediumModified 2 X CHEV + Soil P49 Medium Modified Artificial Seawater MediumPolytomella Medium Modified CHEV Proteose Medium Porphridium Medium SnowAlgae Media Soil + Seawater Medium Soil Extract Medium SS Diatom MediumSoilwater: BAR Medium Soilwater: GR− Medium Soilwater: GR−/NH4 MediumSoilwater: GR+ Medium Soilwater: GR+/NH4 Medium Soilwater: PEA MediumSoilwater: Peat Medium Soilwater: VT Medium Spirulina Medium Tap MediumTrebouxia Medium Volvocacean Medium Volvocacean-3N Medium Volvox MediumVolvox-Dextrose Medium Waris Medium Waris + Soil Extract Medium

A medium suitable for culturing Chlorella protothecoides comprisesProteose Medium. This medium is suitable for axenic cultures, and a 1Lvolume of the medium (pH ˜6.8) can be prepared by addition of 1 g ofproteose peptone to 1 liter of Bristol Medium. Bristol medium comprises2.94 mM NaNO₃, 0.17 mM CaCl₂.2H₂O, 0.3 mM MgSO₄.7H₂O, 0.43 mM, 1.29 mMKH₂PO₄, and 1.43 mM NaCl in an aqueous solution. For 1.5% agar medium,15 g of agar can be added to 1 L of the solution. The solution iscovered and autoclaved, and then stored at a refrigerated temperatureprior to use.

Other suitable media for use with the methods of the invention can bereadily identified by consulting the URL identified above, or byconsulting other organizations that maintain cultures of microorganisms,SAG the Culture Collection of Algae at the University of Göttingen(Göttingen, Germany), CCAP the culture collection of algae and protozoamanaged by the Scottish Association for Marine Science (Scotland, UnitedKingdom), and CCALA the culture collection of algal laboratory at theInstitute of Botany (T{hacek over (r)}ebo{hacek over (n)}, CzechRepublic).

The present methods are particularly suitable for microalgae having ahigh lipid content (e.g., at least 20% lipids by dry weight). Processconditions can be adjusted to increase the percentage weight of cellsthat is lipid. For example, in certain embodiments, a microbe (e.g., amicroalgae) is cultured in the presence of a limiting concentration ofone or more nutrients, such as, for example, nitrogen and/or phosphorousand/or sulfur, while providing an excess of fixed carbon energy such asglucose. Nitrogen limitation tends to increase microbial lipid yieldover microbial lipid yield in a culture in which nitrogen is provided inexcess. In particular embodiments, the increase in lipid yield is fromat least about 10% to 100% to as much as 500% or more. The microbe canbe cultured in the presence of a limiting amount of a nutrient for aportion of the total culture period or for the entire period. Inparticular embodiments, the nutrient concentration is cycled between alimiting concentration and a non-limiting concentration at least twiceduring the total culture period.

To increase lipid as a percentage of dry cell weight, acetate can beemployed in the feedstock for a lipid-producing microbe (e.g., amicroalgae). Acetate feeds directly into the point of metabolism thatinitiates fatty acid synthesis (i.e., acetyl-CoA); thus providingacetate in the culture can increase fatty acid production. Generally,the microbe is cultured in the presence of a sufficient amount ofacetate to increase microbial lipid yield, and/or microbial fatty acidyield, specifically, over microbial lipid (e.g., fatty acid) yield inthe absence of acetate. Acetate feeding is a useful component of themethods provided herein for generating microalgal biomass that has ahigh percentage of dry cell weight as lipid.

In a steady growth state, the cells accumulate oil (lipid) but do notundergo cell division. In one embodiment of the invention, the growthstate is maintained by continuing to provide all components of theoriginal growth media to the cells with the exception of a fixednitrogen source. Cultivating microalgae cells by feeding all nutrientsoriginally provided to the cells except a fixed nitrogen source, such asthrough feeding the cells for an extended period of time, can result ina high percentage of dry cell weight being lipid. In some embodiments,the nutrients, such as trace metals, phosphates, and other components,other than a fixed carbon source, can be provided at a much lowerconcentration than originally provided in the starting fermentation toavoid “overfeeding” the cells with nutrients that will not be used bythe cells, thus reducing costs.

In other embodiments, high lipid (oil) biomass can be generated byfeeding a fixed carbon source to the cells after all fixed nitrogen hasbeen consumed for extended periods of time, such as from at least 8 to16 or more days. In some embodiments, cells are allowed to accumulateoil in the presence of a fixed carbon source and in the absence of afixed nitrogen source for over 30 days. Preferably, microorganisms grownusing conditions described herein and known in the art comprise lipid ina range of from at least about 20% lipid by dry cell weight to about 75%lipid by dry cell weight.

Another tool for allowing cells to accumulate a high percentage of drycell weight as lipid involves feedstock selection. Multiple species ofChlorella and multiple strains within a species of Chlorella accumulatea higher percentage of dry cell weight as lipid when cultured in thepresence of biodiesel glycerol byproduct than when cultured in thepresence of equivalent concentrations of pure reagent grade glycerol.Similarly, Chlorella can accumulate a higher percentage of dry cellweight as lipid when cultured in the presence of an equal concentration(weight percent) mixture of glycerol and glucose than when cultured inthe presence of only glucose.

Another tool for allowing cells to accumulate a high percentage of drycell weight as lipid involves feedstock selection as well as the timingof addition of certain feedstocks. For example, Chlorella can accumulatea higher percentage of dry cell weight as lipid when glycerol is addedto a culture for a first period of time, followed by addition of glucoseand continued culturing for a second period of time, than when the samequantities of glycerol and glucose are added together at the beginningof the fermentation. See PCT Publication No. 2008/151149, incorporatedherein by reference.

The lipid (oil) percentage of dry cell weight in microbial lipidproduction can therefore be improved, at least with respect to certaincells, by the use of certain feedstocks and temporal separation ofcarbon sources, as well as by holding cells in a heterotrophic growthstate in which they accumulate oil but do not undergo cell division. Theexamples below show growing various microbes, including several strainsof microalgae, to accumulate higher levels of lipids as DCW.

In another embodiment, lipid yield is increased by culturing alipid-producing microbe (e.g., microalgae) in the presence of one ormore cofactor(s) for a lipid pathway enzyme (e.g., a fatty acidsynthetic enzyme). Generally, the concentration of the cofactor(s) issufficient to increase microbial lipid (e.g., fatty acid) yield overmicrobial lipid yield in the absence of the cofactor(s). In a particularembodiment, the cofactor(s) are provided to the culture by including inthe culture a microbe (e.g., microalgae) containing an exogenous geneencoding the cofactor(s). Alternatively, cofactor(s) may be provided toa culture by including a microbe (e.g., microalgae) containing anexogenous gene that encodes a protein that participates in the synthesisof the cofactor. In certain embodiments, suitable cofactors include anyvitamin required by a lipid pathway enzyme, such as, for example: biotinor pantothenate. Genes encoding cofactors suitable for use in theinvention or that participate in the synthesis of such cofactors arewell known and can be introduced into microbes (e.g., microalgae), usingconstructs and techniques such as those described herein.

Process conditions can be adjusted to increase the yields of lipidssuitable for multiple uses including, but not limited to, biodiesel.Process conditions can also be adjusted to reduce production cost. Forexample, in certain embodiments, a microbe (e.g., a microalgae) iscultured in the presence of a limiting concentration of one or morenutrients, such as, for example, nitrogen, phosphorus, and/or sulfur.This condition tends to increase microbial lipid yield over microbiallipid yield in a culture in which the nutrient is provided in excess. Inparticular embodiments, the increase in lipid yield is at least about:10% 20 to 500%.

Limiting a nutrient may also tend to reduce the amount of biomassproduced. Therefore, the limiting concentration is typically one thatincreases the percentage yield of lipid for a given biomass but does notunduly reduce total biomass. In exemplary embodiments, biomass isreduced by no more than about 5% to 25%. The microbe can be cultured inthe presence of a limiting amount of nutrient for a portion of the totalculture period or for the entire period. In particular embodiments, thenutrient concentration is cycled between a limiting concentration and anon-limiting concentration at least twice during the total cultureperiod.

The microalgal biomass generated by the culture methods described hereincomprises microalgal oil (lipid) as well as other constituents generatedby the microorganisms or incorporated by the microorganisms from theculture medium during fermentation.

Microalgal biomass with a high percentage of oil/lipid accumulation bydry weight has been generated using different methods of culture knownin the art. Microalgal biomass with a higher percentage of oil/lipidaccumulation is useful in with the methods of the present invention. Liet al. describe Chlorella vulgaris cultures with up to 56.6% lipid bydry cell weight (DCW) in stationary cultures grown under autotrophicconditions using high iron (Fe) concentrations (Li et al., BioresourceTechnology 99(11):4717-22 (2008). Rodolfi et al. describe Nanochloropsissp. and Chaetoceros calcitrans cultures with 60% lipid DCW and 39.8%lipid DCW, respectively, grown in a photobioreactor under nitrogenstarvation conditions (Rodolfi et al., Biotechnology & Bioengineering(2008) [June 18 Epub ahead of print]). Solovchenko et al. describeParietochloris incise cultures with approximately 30% lipid accumulation(DCW) when grown phototropically and under low nitrogen condtions(Solovchenko et al., Journal of Applied Phycology 20:245-251 (2008).Chlorella protothecoides can produce up to 55% lipid (DCW) grown undercertain heterotrophic conditions with nitrogen starvation (Miao and Wu,Bioresource Technology 97:841-846 (2006). Other Chlorella speciesincluding Chlorella emersonii, Chlorella sorokiniana and Chlorellaminutissima have been described to have accumulated up to 63% oil (DCW)when grown in stirred tank bioreactors under low-nitrogen mediaconditions (Illman et al., Enzyme and Microbial Technology 27:631-635(2000). Still higher percent lipid accumulation by dry cell weight havebeen reported, including 70% lipid (DCW) accumulation in Dumaliellatertiolecta cultures grown in increased NaCl conditions (Takagi et al.,Journal of Bioscience and Bioengineering 101(3): 223-226 (2006)) and 75%lipid accumulation in Botryococcus braunii cultures (Banerjee et al.,Critical Reviews in Biotechnology 22(3): 245-279 (2002)).

These and other aspects and embodiments of the invention areillustrated, but not limited, by the examples below; the examples alsohighlight the advantages of the methods of the invention.

IV. Examples

EXAMPLE 1

Cultivation of Microalgae to Achieve High Oil Content

Microalgae strains were cultivated to achieve a high percentage of oilby dry cell weight. Cryopreserved cells were thawed at room temperature,and 500 μl of cells were added to 4.5 ml of medium (4.2 g/L K₂HPO₄, 3.1g/L NaH₂PO₄, 0.24 g/L MgSO₄.7H₂O, 0.25 g/L citric acid monohydrate,0.025 g/L CaCl₂ 2H₂O, 2 g/L yeast extract) plus 2% glucose and grown for7 days at 28° C. with agitation (200 rpm) in a 6-well plate. Dry cellweights were determined by centrifuging 1 ml of culture at 14,000 rpmfor 5 minutes in a pre-weighed Eppendorf tube. The culture supernatantwas discarded and the resulting cell pellet washed with 1 ml ofdeionized water. The culture was again centrifuged, the supernatantdiscarded, and the cell pellets placed at −80° C. until frozen. Sampleswere then lyophilized for 24 hours and dry cell weights were calculated.For determination of total lipid in cultures, 3 ml of culture wasremoved and subjected to analysis using an Ankom system (Ankom Inc.,Macedon, N.Y.) according to the manufacturer's protocol. Samples weresubjected to solvent extraction with an Ankom XT10 extractor accordingto the manufacturer's protocol. Total lipid was determined as thedifference in mass between acid hydrolyzed dried samples and solventextracted, dried samples. Percent oil dry cell weight measurements areshown below in Table 5.

TABLE 5 Cultivation of microalgae to achieve high oil content. SpeciesStrain % Oil SEQ ID NO: Chlorella kessleri UTEX 397 39.42 4 Chlorellakessleri UTEX 2229 54.07 5 Chlorella kessleri UTEX 398 41.67 6Parachlorella kessleri SAG 11.80 37.78 7 Parachlorella kessleri SAG14.82 50.70 8 Parachlorella kessleri SAG 21.11 H9 37.92 9 Protothecastagnora UTEX 327 13.14 10 Prototheca moriformis UTEX 1441 18.02 11Prototheca moriformis UTEX 1435 27.17 12 Chlorella minutissima UTEX 234131.39 13 Chlorella protothecoides UTEX 250 34.24 1 Chlorellaprotothecoides UTEX 25 40.00 2 Chlorella protothecoides CCAP 211/8D47.56 3 Chlorella sp. UTEX 2068 45.32 14 Chlorella sp. CCAP 211/92 46.5115 Chlorella sorokiniana SAG 211.40B 46.67 16 Parachlorella beijerinkiiSAG 2046 30.98 17 Chlorella luteoviridis SAG 2203 37.88 18 Chlorellavulgaris CCAP 211/11K 35.85 19 Chlorella reisiglii CCAP 11/8 31.17 20Chlorella ellipsoidea CCAP 211/42 32.93 21 Chlorella saccharophila CCAP211/31 34.84 22 Chlorella saccharophila CCAP 211/32 30.51 23Culturing Chlorella protothecoides to Achieve High Oil Content

Three fermentation processes were performed with three different mediaformulations with the goal of generating algal biomass with high oilcontent. The first formulation (Media 1) was based on medium describedin Wu et al. (1994 Science in China, vol. 37, No. 3, pp. 326-335) andconsisted of per liter: KH₂PO₄, 0.7 g; K₂HPO₄, 0.3 g; MgSO₄-7H₂O, 0.3 g;FeSO₄-7H₂O, 3 mg; thiamine hydrochloride, 10 μg; glucose, 20 g; glycine,0.1 g; H₃BO₃, 2.9 mg; MnCl₂-4H₂O, 1.8 mg; ZnSO₄-7H₂O, 220 μg;CuSO₄-5H₂O, 80 μg; and NaMoO₄-2H₂O, 22.9 mg. The second medium (Media 2)was derived from the flask media described in Example 1 and consisted ofper liter: K₂HPO₄, 4.2 g; NaH₂PO₄, 3.1 g; MgSO₄-7H₂O, 0.24 g; citricacid monohydrate, 0.25 g; calcium chloride dehydrate, 25 mg; glucose, 20g; yeast extract, 2 g. The third medium (Media 3) was a hybrid andconsisted of per liter: K₂HPO₄, 4.2 g; NaH₂PO₄, 3.1 g; MgSO₄-7H₂O, 0.24g; citric acid monohydrate, 0.25 g; calcium chloride dehydrate, 25 mg;glucose, 20 g; yeast extract, 2 g; H₃BO₃, 2.9 mg; MnCl₂-4H₂O, 1.8 mg;ZnSO₄-7H₂O, 220 μg; CuSO₄-5H₂O, 80 μg; and NaMoO₄-2H₂O, 22.9 mg. Allthree media formulations were prepared and autoclave sterilized in labscale fermentor vessels for 30 minutes at 121° C. Sterile glucose wasadded to each vessel following cool down post autoclave sterilization.

Inoculum for each fermentor was Chlorella protothecoides (UTEX 250),prepared in two flask stages using the medium and temperature conditionsof the fermentor inoculated. Each fermentor was inoculated with 10%(v/v) mid-log culture. The three lab scale fermentors were held at 28°C. for the duration of the experiment. The microalgal cell growth inMedia 1 was also evaluated at a temperature of 23° C. For all fermentorevaluations, pH was maintained at 6.6-6.8, agitations at 500 rpm, andairflow at 1 vvm. Fermentation cultures were cultivated for 11 days.Biomass accumulation was measured by optical density at 750 nm and drycell weight.

Lipid/oil concentration was determined using direct transesterificationwith standard gas chromatography methods. Briefly, samples offermentation broth with biomass was blotted onto blotting paper andtransferred to centrifuge tubes and dried in a vacuum oven at 65-70° C.for 1 hour. When the samples were dried, 2 mL of 5% H₂SO₄ in methanolwas added to the tubes. The tubes were then heated on a heat block at65-70° C. for 3.5 hours, while being vortexed and sonicatedintermittently. 2 ml of heptane was then added and the tubes were shakenvigorously. 2Ml of 6% K₂CO₃ was added and the tubes were shakenvigorously to mix and then centrifuged at 800 rpm for 2 minutes. Thesupernatant was then transferred to GC vials containing Na₂SO₄ dryingagent and ran using standard gas chromatography methods. Percentoil/lipid was based on a dry cell weight basis. The dry cell weights forcells grown using: Media 1 at 23° C. was 9.4 g/L; Media 1 at 28° C. was1.0 g/L, Media 2 at 28° C. was 21.2 g/L; and Media 3 at 28° C. was 21.5g/L. The lipid/oil concentration for cells grown using: Media 1 at 23°C. was 3 g/L; Media 1 at 28° C. was 0.4 g/L; Media 2 at 28° C. was 18g/L; and Media 3 at 28° C. was 19 g/L. The percent oil based on dry cellweight for cells grown using: Media 1 at 23° C. was 32%; Media 1 at 28°C. was 40%; Media 2 at 28° C. was 85%; and Media 3 at 28° C. was 88%.The lipid profiles (in area %, after normalizing to the internalstandard) for algal biomass generated using the three different mediaformulations at 28° C. are summarized below in Table 6.

TABLE 6 Lipid profiles for Chlorella protothecoides grown underdifferent media conditions. Media 1 28° C. Media 2 28° C. Media 3 28° C.(in Area %) (in Area %) (in Area %) C14:0 1.40 0.85 0.72 C16:0 8.71 7.757.43 C16:1 — 0.18 0.17 C17:0 — 0.16 0.15 C17:1 — 0.15 0.15 C18:0 3.773.66 4.25 C18:1 73.39 72.72 73.83 C18:2 11.23 12.82 11.41 C18:3 alpha1.50 0.90 1.02 C20:0 — 0.33 0.37 C20:1 — 0.10 0.39 C20:1 — 0.25 — C22:0— 0.13 0.11Culturing Oleaginous Yeast to Achieve High Oil Content

Yeast strain Rhodotorula glutinis (DSMZ-DSM 70398) was obtained from theDeutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (GermanCollection of Microorganism and Cell Culture, Inhoffenstraβe 7B, 38124Braunschweig, Germany. Cryopreserved cells were thawed and added to 50mL YPD media (described above) with 1× DAS vitamin solution (1000×: 9g/L tricine; 0.67 g/L thiamine-HCl; 0.01 g/L d-biotin; 0.008cyannocobalamin; 0.02 calcium pantothenate; and 0.04 g/L p-Aminobenzoicacid) and grown at 30° C. with 200 rpm agitation for 18-24 hours untilan OD reading was over 5 OD (A600). The culture was then transferred to7-L fermentors and switched to YP1 medium (8.5 g/L Difco Yeast NitrogenBase without Amino Acids and Ammonium Sulfate, 3 g/L Ammonium Sulfate, 4g/L yeast extract) with lx DAS vitamin solution. The cultures weresampled twice per day and assayed for OD (A600), dry cell weight (DCW)and lipid concentration. When the cultures reached over 50 g/L DCW, thecultures were harvested. Based on dry cell weight, the yeast biomasscontained approximately 50% oil. Two samples of yeast biomass weresubjected to direct transesterification and analyzed via GC/FID for alipid profile. The results are expressed in Area Percent, and shown inTable 7, below.

TABLE 7 Lipid profile of transesterified yeast biomass samples. C10:0C12:0 C14:0 C15:0 C16:0 C16:1 C17:0 C18:0 C18:1 C18:2 C18:3α ≧C:20Sample 1 0.03 0.21 3.36 0.25 33.26 0.76 0.20 6.88 42.68 9.28 1.33 1.1Sample 2 0.02 0.10 2.18 0.12 29.94 0.49 0.16 8.17 48.12 7.88 0.84 1.45Cultivation of Rhodococcus opacus to Achieve High Oil Content

A seed culture of Rhodococcus opacus PD630 (DSM 44193, Deutsche Sammlungvon Mikroorganismen und Zellkuttwen GmbH) was generated using 2 ml of acryo-preserved stock inoculated into 50 ml of MSM media with 4% sucrose(see Schlegel, et al., (1961) Arch Mikrobiol 38, 209-22) in a 250 mlbaffle flask. The seed culture was grown at 30° C. with 200 rpmagitation until it reached an optical density of 1.16 at 600 nm. 10 mlof the seed flask was used to inoculate cultures for lipid productionunder two different nitrogen conditions: 10 mM NH₄Cl and 18.7 mM NH₄Cl(each in duplicate). The growth cultures were grown at 30° C. with 200rpm agitation for 6 days. Cells grown in the 10 mM NH₄Cl conditionreached a maximal 57.2% (average) lipid by DCW after 6 days of culture.Cells grown in the 18.7 mM NH₄Cl condition reached a maximal 51.8%(average) lipid by DCW after 5 days in culture.

A sample of Rhodococcus opacus biomass was subjected to directtransesterification and analyzed via GC/FID for a lipid profile. Theresults were: C14:0 (2.33); C15:0 (9.08); C16:0 (24.56); C16:1 (11.07);C17:0 (10.50); 2 double bond equivalent (2DBE) C17 species (19.90);C18:0 (2.49); C18:1 (17.41); C18:2 (0.05); C19:0 (0.75) and 2DBE C19species (1.87).

EXAMPLE 2

Diversity of Lipid Chains in Microalgal Species

Lipid samples from a subset of strains grown in Example 1, and listed inTable 5, were analyzed for lipid profile using HPLC. Results are shownbelow in Table 8.

TABLE 8 Diversity of lipid chains in microalgal species. MicroalgalStrain C:14:0 C:16:0 C:16:1 C:18:0 C:18:1 C:18:2 C:18:3 C:20:0 C:20:1 C.protothecoides 0.57 10.30 0 3.77 70.52 14.24 1.45 0.27 0 (UTEX 250) C.protothecoides 0.61 8.70 0.30 2.42 71.98 14.21 1.15 0.20 0.24 (UTEX 25)C. kessleri (UTEX 0.68 9.82 0 2.83 65.78 12.94 1.46 0 0 397) C. kessleri(UTEX 1.47 21.96 0 4.35 22.64 9.58 5.2 3.88 3.3 2229) Prototheca 0 12.010 0 50.33 17.14 0 0 0 stagnora (UTEX 327) Prototheca 1.41 29.44 0.703.05 57.72 12.37 0.97 0.33 0 moriformis (UTEX 1441) Prototheca 1.0925.77 0 2.75 54.01 11.90 2.44 0 0 moriformis (UTEX 1435)

EXAMPLE 3

Drum Drying Microalgal Biomass

An F-tank batch of Chlorella protothecoides (UTEX 250) (about 1,200gallons) was used to generate biomass for extraction processes. Thebatch was allowed to run for approximately 100 hours, while controllingthe glucose levels at 16 g/L, after which time the corn syrup feed wasterminated. Residual glucose levels dropped to approximately 0 g/L twohours later. The final broth volume was 1,120 gallons. Both in-processcontamination checks and a thorough analysis of a final broth samplefailed to show any signs of contamination. The microalgal biomasscontained 38% oil based on dry cell weight (DCW).

The microalgal biomass was then dried on an atmospheric double drumdryer. The broth was fed through a nozzle onto the two steam-heateddrums that were counter-rotating toward each other. The broth wasmechanically spread by the action of the counter-rotating drums intothin split sheets on both hot cylinders. The adhering thin sheets ofbroth were rapidly dried conductively by the high heat flux of thecondensing steam inside the drums. The steam pressure ranged from 45 to105 psig and the drum rotational speed was adjusted to between 2 to 20rpm. The moisture content of the biomass after drum drying was between3-10% (by weight).

Heat-conditioning Microbial Biomass

Chlorella protothecoides was produced and drum dried using the methodsdescribed in Example 6. Drum dried microalgal biomass was heatconditioned using a 4-deck vertical stacked conditioner (model 424,French Oil Mill Machinery, Piqua, Ohio). Each deck held up to 2.8 cubicfeet of material. The vertical stacked conditioner was preheated using45 to 100 psig steam for about an hour before heat conditioning of thebiomass. After pre-heating, the microalgal biomass was loaded onto thetop deck of the vertical stacked conditioner and guided to a chuteleading to the next deck below by a sweeper arm on each deck, which wasmounted to a common vertical shaft powered by an electric motor. A 150pound load of drum dried microalgal biomass filled the verticalconditioner, and in order to ensure uniform heat-conditioning, thebiomass was circulated by opening the vertical stacked conditioner'sbottom discharge gate valve and returning the biomass to the top of thevertical conditioner. The biomass temperature in each deck was monitoredand controlled between 180 and 250° F. in order to prevent scorching.Heat-conditioning residence times were varied from 10 to 60 minutes toobtain biomass of varying moisture content. The heat-conditioned biomasswas unloaded into covered polyethylene carts and immediately pressed inan oilseed press. Samples of biomass before and after heat-conditioningwere analyzed for moisture and oil content.

Oil Extraction from Microalgae using a Benchtop Taby Pressen

Drum dried Chlorella protothecoides (UTEX 250) biomass made according tothe methods above was dried such that the resulting moisture content wasabout 5-5.5%. The microalgal biomass contained 48.5% oil based on drycell weight (DCW). The biomass was fed through a Taby Pressen Type 70oil press with a 2.2 Hp motor and 70 mm screw diameter. The press waspreheated to a barrel temperature of 100° C. No oil was extracted underthese conditions. Another press run was performed using the same lot ofdrum-dried biomass with the moisture adjusted down to 0.5% moisture byweight using a forced air oven at 70° C. for 30 minutes as aconditioning step prior to feeding it into the press. The press barrelwas heated to 82° C. and the drum-dried, conditioned microalgal biomasswas fed through the bench-top press. Approximately 68% of the availableoil (by weight) was recovered and the pressed cake, or spent biomass,was then solvent extracted to recover the residual oil. After multipleexperiments with microalgal biomass of varying oil content (between40-55% oil DCW), the pressed cake had approximately 30% oil as measuredby analytical methods every time.

The analytical method for determining percent lipid/oil in a sample wasbased on a modified Soxlet method. Briefly, 1 gram of sample was weighedout and subjected to acid hydrolysis followed by petroleum ether solventextraction. Both acid hydrolysis and petroleum either extraction wasaccelerated by heat using the MARS Microwave accelerated reactionsystem. The petroleum ether solvent was then evaporated and the amountof extracted lipid was determined gravimetrically.

Small Scale Solvent Extraction

Pressed cake generated from the bench top press was solvent extracted torecover the residual oil. Excess petroleum ether was added to thepressed cake (5:1 weight by volume) and mixed for a minimum of an hourat room temperature. The petroleum ether mixture was then passed througha Buchner funnel containing a 5 μm filter. The solids were collectedfrom the filter and then subjected to 3 additional washes with petroleumether of 2× volume each. The filtered petroleum ether mixture and washeswere pooled and placed in a RotoVap (2 L) to distill the petroleumether. The remaining oil was collected and weighed. Upon microscopicinspection of the petroleum ether extracted cake, there was no free oildetected in the cake after petroleum ether extraction. However, lipidvesicles were still seen in intact (unbroken) algae cells. This methodwill recover 100% of the free oil in the pressed cake, but not in intactalgae cells. This small scale solvent extraction process can be used todetermine the effectiveness of an expeller press at breaking or crackingthe algae cells.

EXAMPLE 4

Oil Extraction from Microalgae Using a Lab Scale Komet Press

Thirty (30) kilograms of drum dried Chlorella protothecoides (UTEX 250)microalgal biomass containing about 48% oil by DCW with moisture contentof about 5% was run through a Komet oilseed press with a 65 mm diameterin a pre-press conditioning step with the discharge cone completelydisengaged. Under this low pressure pre-press condition, no oil wasreleased; however, the dried microalgal biomass was converted from looseflakes into pre-pressed pellets. FIG. 2a shows the drum-dried biomassmaterial that was fed into the press. FIG. 2b shows the pre-pressedpellets. The pre-pressed pellets were collected and run through the samepress under full press conditions with the discharge cone completelyengaged for maximum pressure. The result was a 69% recovery of the oilfrom the pellets.

Spent cake from the press was then subjected to solvent extraction usingiso-hexane in a percolation type extractor. The iso-hexane extractionyielded an additional 1 kg of oil. The combination of the conditionedpressing followed by the hexane extraction recovered a total of 76% ofthe total available oil from the dried microalgal biomass. These resultsare summarized in Table 9 below.

TABLE 9 Summary of results from Komet press run. Total Biomass 30kilograms Percent Oil (DCW) 48% Total Available Oil by Weight 14.4kilograms Recovered Virgin Oil (Conditioned 10 kilograms pressing) % OilRecovery from Pressing 69% Available Oil by Weight After Pressing 4.4kilograms % Oil Recovery from Hexane Extraction 23% of available oilfrom the pressed cake (approx. 1 kilogram oil) Total Percentage of OilRecovered 76%

EXAMPLE 5

Pilot-scale Pressing of Microalgae Using a Press Aid

Microalgal biomass (Chlorella protothecoides UTEX 250) containing 38%oil by DCW was dried using a drum dryer with a resulting moisturecontent of about 3.5% (as measured by a moisture analyzer). An L-250(3.5″ diameter) French pilot scale oilseed screw press (French Oil MillMachinery Company, Piqua, Ohio) was used in the following experiments.The core main barrel (or cage) had a diameter of 3.5 inches. The screwconsisted of alternating worms and collars set up for a compressionratio of 7 to 18. The main drive was powered by a 20 horsepower electricmotor and the main shaft rotational speed was 20 rpm. The cage and shaftwas preheated to between 180° F. and 260° F. by using indirect steam. Acone gap of 0.5 inches, measured between cone bracket and cone mountingplate was used. The cone gap was adjusted by using the 3-positiondirectional valves and the hand pump of the hydraulic cone cylinder.

Dried switchgrass was used as a press aid for microalgae. Drum-driedalgal biomass of 38% oil DCW was mixed with switchgrass to form a 20%switchgrass/biomass, 5% switchgrass/biomass, or biomass only samples.All three algal biomass samples were separately heat conditioned in avertical stacked conditioner (as described above) for 30 minutes at 121°C. The press was heated to a barrel temperature of 93° C. prior to theaddition of the biomass. Approximately 39.5% of the total available oil(by weight) was recovered from pressing the 20% switchgrass/biomass andalso yielded a good quality pressed cake for hexane extraction.Approximately 25% of the total available oil (by weight was recoveredfrom pressing the 5% switchgrass/biomass and also yielded a good qualitypressed cake for hexane extraction. The biomass only condition yieldedlower oil recovery, about 5% of the total oil (by weight), and thepressed cake was of lower quality for hexane extraction.

Soybean hulls were also used as a press aid with microalgae. Drum-driedalgal biomass of 38% oil DCW was mixed with soybean hulls to form a 20%soybean hull/biomass, 10% soybean hull/biomass or biomass only samples.All three algal biomass samples were separately heat conditioned in avertical stacked conditioner for 30 minutes at 121° C. The press washeated to a barrel temperature of 93° C. prior to the addition of thebiomass. The 20% soybean hull/biomass mixture did not feed through thepress; therefore, no oil was recovered. Approximately 22.5% of the totalavailable oil (by weight) was recovered from pressing the 10% soybeanhull/biomass and also yielded a good quality pressed cake for hexaneextraction. The biomass only condition yielded no oil and clogged thescrew assembly of the expeller press. In the 5%, 20% switchgrass and 10%soybean hulls conditions, there were about 25% solids by weight in theoil recovered. The results are summarized in Table 10 below, which showthe total percent oil recovered by weight with the weight of the solidssubtracted out.

TABLE 10 Pressing microalgae with use of press aids. Percent % oilQuality of pressed cake Press Aid added recovered for solvent extractionSwitchgrass 0   ~5% Poor Switchgrass  5% 24.9% Good Switchgrass 20%39.5% Good Soybean hulls 0 none N/A Soybean hulls 10% 22.5% Good Soybeanhulls 20% none N/A

EXAMPLE 6

Effects of Moisture Content on Oil Recovery

Chlorella protothecoides (UTEX 250) algal biomass of 38% oil DCW wasdrum dried according to the methods described in Example 3. The moisturecontent was measured at approximately 3 to 5% and was not conditionedprior to feeding into the screw press. 72 pounds of the biomass was fedinto 3.5″ oil seed screw press (French Oil Mill Company, Piqua, OH)preheated to 200° F. Heavy footing or solids was observed to be pushedbetween the bars of the cage throughout all sections of the press.Approximately 5% oil was recovered (after solids were removed). Solventextraction of the pressed cake recovered an additional 58% of the totalavailable oil (approximately 7 kgs). Analysis of the solvent extractedcake showed that there was approximately 18.6% residual oil. Totalrecovery (press and solvent extraction) of oil was 62%, indicating thatthe press only broke or lysed 62% of the microalgal cells. The poor oilrecovery (5%) and the heavy footing that was observed indicated that theconditions (e.g., moisture content of the microbial biomass) were notoptimal.

A series of tests were performed to establish an optimal range ofmoisture content for dried microbial biomass that would yield thehighest recovery of oil in an expeller press. Microalgal biomasscontaining 51.3% oil by DCW was dried using a drum dryer. A French 3.5″oilseed press (comparable to the L250 French press) was used and thesetup was identical to that described in Example 5. The dried algalbiomass was heat-conditioned in a vertical stacked conditioner at 250°F. The time of heat-conditioning was varied to achieve the differentmoisture content levels. The press barrel was pre-heated to andmaintained at 200° F. during all of the experiments, unless otherwisenoted. The heat-conditioned microalgal biomass was introduced slowlyinto the oilseed press, while monitoring the press electrical load. Thefeed rate was controlled by a variable-speed screw conveyor. The feedrate was determined by adding the weights of crude oil and pressed cakecollected over 2-3 minutes. Samples of each of the pressed cake from thedifferent conditions were analyzed using analytical methods describedabove to measure lipid/oil content.

A second set of experiments was done with the same lot of drum-driedmicroalgal biomass as above. One batch of microalgal biomass wasconditioned in the vertical stacked conditioner at 200° F. and had afinal moisture content of 1.2%. Another batch of microalgal biomass wasconditioned in the vertical stacked conditioner at 290° F. and had afinal moisture content of 0.7%. Both batches were pressed in the L250,3.5″ diameter oilseed press with the barrel temperature at 200° F. Thebatch with the 1.2% moisture content yielded 54.3% oil recovery (byweight), which is comparable to the results from the above batch of 1.2%moisture content biomass, which yielded 48.2% oil recovery. The 0.7%moisture content condition yielded the best results, producing a goodquality cake for subsequent solvent extraction and a yield of 73% oilrecovery. However, the oil was slightly darker than the oil producedfrom the biomass that was conditioned at 200° F. Table 11 belowsummarizes the test moisture content and percent oil recovered (aftersubtracting out the solids) from these two sets of experiments.

TABLE 11 Effects of moisture content on oil recovery. % oil in OilingQuality of pressed % oil pressed rate cake for Moisture contentrecovered cake (g/min) solvent extraction 0.2% 49.1% 8% 355 Poor-burnt;powdery 1% 66.9% 29% 596 Good 1.2% 48.2 33% 518 Good 2.4% 34.9% 39% 277Good 1.2%; conditioning 54.3% 32% 520 Good temp 200° F. 0.7%;conditioning   73% 16% 697 Good; Oil was temp 290° F. slightly darkerthan 200° F. runs

The results from these two sets of experiments indicated that theoptimum moisture content to achieve the highest percent oil recovery anda good quality pressed cake for subsequent solvent extraction in thisExample is between 0.7% and 1.2%. This range of moisture content in themicroalgal biomass also produced less solids in the oil (less than 25%by weight). Other similar experiments showed that a moisture contentbelow 0.5% resulted in heavy footing (solids) in the oil (over 40% byweight), which impacts overall yield, and produced a burnt, powdery,poor quality cake that was not suitable for subsequent solventextraction (See FIG. 2a ). At a moisture content higher than 1.2%, theoverall yield of oil was lower, but the pressed cake produced was ofvery good quality for subsequent solvent extraction (See FIG. 2b ). Thisresult indicates that moisture content plays a major role in the qualityand texture of the pressed cake from microbial biomass, and there is aninhibition of percent oil yield if the moisture content is too high.

EXAMPLE 7

Prototheca in Lab Press with Lipid Profile of Pressed, Extracted, andCombined Materials

Approximately 2 kg of drum-dried Prototheca moriformis algal biomasswith 60% lipid DCW was pressed using a bench-top Taby Type 70 press (seeExample 8 above for press conditions). The dried biomass was heatconditioned in a forced air oven at 212° F. for 30 minutes. The presswas preheated to 200° F. and 235.5 grams of oil was recovered (19%),after solids were removed. The oil was collected and analyzed for lipidprofile (expressed as area %), chlorophyll and carotenoid content usingstandard methods. The residual oil in the pressed cake was recovered viaa batch extraction with petroleum ether (as described in Example 3). Atotal of 311 grams of oil was extracted out of 961 grams of pressedcake. The extracted oil was also analyzed for lipid profile (expressedas area %), chlorophyll and carotenoids, which are summarized below.Overall, the lipid profile, chlorophyll and carotenoid content frompressed oil and solvent extracted oil were very similar.

TABLE 12 Lipid profile of oil extracted from Prototheca moriformis.Pressed oil (Area %) Solvent extracted oil (Area %) C12:0 0.05 0.05C14:0 1.36 1.37 C14:1 0.02 0.02 C15:0 0.04 0.04 C16:0 19.90 20.11 C16:10.85 0.85 C18:0 4.11 4.15 C18:1 64.81 64.56 C18:2 7.83 7.83 C20:0 0.030.03

TABLE 13 Carotenoid and chlorophyll content in oil extracted fromPrototheca morifomis. Pressed oil Solvent extracted oil (mcg/ml)(mcg/ml) cis-Lutein 0.041 0.042 trans-Lutein 0.140 0.112trans-Zeaxanthin 0.045 0.039 cis-Zeaxanthin 0.007 0.013t-alpha-Cryptoxanthin 0.007 0.010 t-beta-Cryptoxanthin 0.009 0.010t-alpha-Carotene 0.003 0.001 c-alpha-Carotene none detected nonedetected t-beta-Carotene 0.010 0.009 9-cis-beta-Carotene 0.004 0.002Lycopene none detected none detected Total Carotenoids 0.267 0.238Chlorophyll <0.01 mg/kg <0.01 mg/kg

Additionally, elemental analysis was also performed on pressed oil andsolvent extracted oil from Prototheca moriformis using ICP massspectrometry. The results are summarized below in Table 14.

TABLE 14 Elemental analysis of oil extracted from Prototheca moriformis.Solvent extracted Pressed oil (ppm) oil (ppm) Lithium <2 <2 Beryllium <2<2 Boron <2 <2 Sodium 12 12 Magnesium <2 2 Aluminum 8 8 Phosphorus <2 <2Potassium 12 26 Calcium 11 <2 Scandium <2 <2 Titanium <2 <2 Vanadium <2<2 Chromium <2 <2 Manganese <2 <2 Iron <2 <2 Cobalt <2 <2 Nickel <2 <2Copper <2 <2 Zinc <2 <2 Gallium <2 <2 Germanium <2 <2 Arsenic <2 <2Selenium <2 <2 Rubidium <2 <2 Strontium <2 <2 Yttrium <2 <2 Zirconium <2<2 Niobium <2 <2 Molybdenum <2 <2 Ruthenium <2 <2 Rhodium <2 <2Palladium <2 <2 Silver <2 <2 Cadmium <2 <2 Indium <2 <2 Tin <2 <2Antimony <2 <2 Tellurium <2 <2 Cesium <2 <2 Barium <2 <2 Lanthanum <2 <2Cerium <2 <2 Praseodymium <2 <2 Neodymium <2 <2 Samarium <2 <2 Europium<2 <2 Gadolinium <2 <2 Terbium <2 <2 Dysprosium <2 <2 Holmium <2 <2Erbium <2 <2 Thulium <2 <2 Ytterbium <2 <2 Lutetium <2 <2 Hafnium <2 <2Tantalum <2 <2 Tungsten <2 <2 Rhenium <2 <2 Iridium <2 <2 Platinum <2 <2Gold <2 <2 Thallium <2 <2 Lead <2 <2 Bismuth <2 <2 Thorium <2 <2 Uranium<2 <2 Iodine 28 <4 Sulfur (ICP) <5 <5 Mercury <2 <2 Chloride <8 <6

EXAMPLE 8

Pilot Scale Pressing of Prototheca moriformis

Prototheca moriformis (UTEX 1435) containing approximately 66% oil (bydry cell weight) was drum dried using the methods described in Example3. After drum drying, the moisture content of the biomass was about2.7%. The dried biomass was then heat-conditioned using methodsdescribed in Example 3. The moisture content of the biomass afterheat-conditioning was approximately 0.6-1.4%. The algal biomass was thenfed into a 3.5″ oil seed screw press (French Oil Mill Company, PiquaOhio) with the cage preheated to195-220° F. Heavy footing was observedto be pushed between the bars of the cage, indicating that theconditions were not optimal. Approximately 47.9% oil (based on weightand theoretical calculation of available oil in the biomass) wasrecovered with fines (solids). The solids were removed by centrifugationand the total oil yield was 31.9% after clarification. Analysis of thepressed cake showed that there was approximately 22% (by weight of thepressed cake) residual oil. Another batch of biomass that was similarlyprepared was also run through the press with similar yield in oilrecovery (57.3% including solids in the oil).

EXAMPLE 9

Use of Press Aids with Oil Extraction from Prototheca moriformis

A series of tests were performed with different press aids on thelab-scale bench top Taby press in order to see if the addition of apress aid can increase oil yields. Different press aids were added tothe fermentation broth after harvesting the biomass. The pressaid/biomass were then dried together on a drum dryer and thenheat-conditioned. This press aid/biomass mixture was then fed into alab-scale Taby press under conditions described above in Example 3. Acondition with no added press aid was used as a negative control.

For the negative control, 3L of fermentation broth containing biomasswith 63.4% oil was dried on a drum dryer. The dried biomass was fedthrough the bench top press and yielded minimal amount of oil. The nextcondition was 3 L of fermentation broth with 150 g (5% by wet weight ofthe fermentation broth) cellulosic filter aid (PB20, EP Minerals,Nevada). The mixture was then dried on a drum dryer and had a moisturecontent of 2.25% after drum drying. The cellulose/biomass was then heatconditioned in a 110° C. oven for 20-30 minutes, to a final moisturecontent of 1.2%. This conditioned cellulose/biomass was then fed intothe lab scale press. Based on theoretical calculations of available oil,there was approximately 145 grams of available oil in the biomass.Approximately 148 grams of oil was recovered using the lab scale press,making total oil recovery about 100%.

The next conditioned tested was the addition of 5% coarse-ground soyhulls (by wet weight). 150 grams of soy hulls was mixed with 3 L offermentation broth containing Prototheca moriformis biomass. During themixing process, it was observed that the coarse-ground soy hulls tendedto settle out of solution, so constant mixing was required. The mixturewas then dried on the drum dryer and had a moisture content of 6.5%after drum drying. The dried soyhull/biomass was then heat conditionedin a 110° C. oven for 20-30 minutes, to a final moisture content of2.5%. The heat-conditioned soyhull/biomass was then fed through the labscale screw press. Out of the calculated 162 grams of available oil, 46grams of oil was recovered from the lab scale screw press, making totaloil recovery at 28%. These experiments were repeated with another lot ofPrototheca moriformis fermentation broth and 2% coarse ground soyhullsadded, 1% soyhulls added, 2% cellulose added and 1% cellulose added. Anegative control with just drum dried fermentation broth was alsotested. Minimal amount of oil was recovered from the negative control.From the 2% soyhull condition, 42% of the available oil was recoveredand 30% of the available oil was recovered from the 1% soyhullcondition. From the 2% cellulose condition, 40% of the available oil wasrecovered and 10% of the available oil was recovered from the 1%cellulose condition.

Additional experiments were performed using finely ground soy hulls anddry back addition to the fermentation broth as press aids. Because thecourse ground soy hulls have the tendency to settle out, the courseground soy hulls were finely ground using a coffee grinder on the finestsetting. After grinding, the finely ground soy hulls had a powderytexture. Dryback (2× pressed cake of Prototheca moriformis biomasshaving 5% oil content) was also ground up for this experiment. Thecontrol condition was Prototheca moriformis biomass with approximately60% oil, without the addition of press aid . 150 grams of either finelyground soy hulls (5%) or ground dry back (5%) was added to 3 L offermentation broth containing Prototheca moriformis biomass. The algalbiomass and press aid mixture was then dried on a drum dryer. Thecontrol (Prototheca moriformis biomass only) condition had a moisturecontent of 4% after drum drying, the 5% finely ground soy hull conditionhad a moisture content of 2.3% after drum drying, and the 5% dry backcondition had a moisture content of 2.5% after drum drying. Each of thebiomass was then dried in a 110° C. oven for 20-30 minutes in order toheat condition the biomass. The final moisture content for the controlbiomass was 2%, the final moisture content for the 5% finely ground soyhull addition was 1.3% and the final moisture content for the 5% dryback addition was 1.01%. After heat conditioning, each of the biomasswas then fed through a lab-scale Taby screw press under conditionsdescribed above in Example 3. The extracted oil and the pressed cake wascollected and analyzed for an estimated yield. For the controlcondition, 6.7 grams of oil was collected (after removal of solids fromthe oil), for an approximate yield of 2.8%. Heavy footing was observedthrough out the press and the pressed cake clogged the discharge end ofthe press. In the 5% finely ground soy hull added condition, 148.2 gramsof oil was collected (after removal of solids from the oil), for anapproximate yield of 79.2% recovery. There was minimal amount of footingduring pressing and very low amount of solids were in the pressed oilbefore clarification. In the 5% dryback added condition, 195.9 g of oilwas collected (after removal of solids from the oil), for an approximateyield of 54.6%. There was minimal amount of footing during pressing andvery small amount of solids were in the pressed oil beforeclarification. These results are consistent with the above results,where the addition of press aids to the fermentation broth (containingmicroalgae biomass) followed by co-drying on a drum drier producedbiomass that had an increased oil yield when pressed on a screw press ascompared to microalgal biomass with no press aid added.

Pilot Scale Pressing of Prototheca moriformis Using a Press Aid

Pilot plant scale trial were performed to evaluate ground soy hulls aspress aids when dry mixing with drum dried Prototheca moriformismicroalgal biomass. Ground soy hulls were mixed with drum dried biomassat 10 and 20% w/w based on finished weight of the mix. Then themicroalgal biomass/soy hull mix was heat conditioned in a French 424vertical stacked conditioner before being pressed in a 3.5″ screw press(French Oil Mill Company, Piqua, Ohio). Oil and pressed cake wererecovered and weighed to estimate yields.

A control batch of Prototheca moriformis biomass was prepared in asimilar manner but without the inclusion of soyhulls. The control batchhad an oil content of 52% (DCW) and a moisture content of 2.57% afterdrum drying. 70 pounds of the control batch was heat conditioned for 30minutes in a vertical stacked conditioner at 195-223° F. and themoisture content was reduced to 0.81-0.95%. 72 pounds of 10% soy hullsadded biomass with an initial moisture content of 3.30% was conditionedat 195-223.5° F. for 30 minutes and the moisture content was reduced to1.06%. 70 pounds of the 20% soy hulls added biomass with an initialmoisture content of 3.07% was heat conditioned at 208-227° F. for 30minutes and the moisture content was reduced to 1.47%. The heatconditioned biomass was then fed into the screw press. In the controlbatch, 30 pounds (out of the 70 pounds that was heat conditioned) wasfed through the press before the press clogged. Approximately 4.0 poundsof oil was recovered (including solids) from the 30 pounds of biomassthat was fed through, making the yield approximately 20.5%. In the 10%soy hull condition, 61 pounds (out of the 72 pounds that was heatconditioned) was fed through the press and approximately 7.0 pound ofoil was recovered (including solids), making the yield approximately20%. In the 20% soy hulls test, all 70 pounds of the heat conditionedmaterial was fed through the press, but minimal (unmeasured) amounts ofoil was recovered.

With the success of the lab scale press experiments described above, theaddition of press aids to the fermentation broth after harvesting thealgal biomass was scaled up for a pilot scale oil screw press (3.5″ oilseed screw press (French Oil Mill Company, Piqua, Ohio)). Protothecamoriformis biomass was prepared under three different experimentalconditions: a negative control with no cellulose (PB20) added, biomasswith 25 g/L cellulose added to the fermentation broth after harvesting,and biomass with 50 g/L cellulose added to the fermentation broth afterharvesting. Biomass from all three conditions was dried on a drum dryer.The negative control biomass had about 58% oil content (DCW) and amoisture content of 6.68%. 140 pounds of the negative control biomasswas then conditioned in a vertical stacked conditioner at 225° F. for 45minutes and the moisture content after heat-conditioning was 2.5-3.5%The 25 g/L cellulose added biomass had a moisture content of 5.30% afterdrum drying. 200 pounds of the 25 g/L cellulose added biomass was thenconditioned in a vertical stacked conditioner at 200° F. for 45 minutesand the moisture content after heat-conditioning was 2.5-3.5%. The 50g/L cellulose added biomass had a moisture content of 4.35% after drumdrying. 115 pounds of the 50 g/L cellulose added biomass was thenconditioned in a vertical stacked conditioner at 200° F. for 45 minutesand the moisture content after heat-conditioning was 2.5-3.5%. Biomassfrom each experimental condition was then fed through the 3.5″ oil seedpress. In the negative control condition, 32.3 pounds of oil (includingsolids or fines in the oil) was recovered. The pressed cake from thenegative control condition was analyzed for residual oil content and thecake contained 42-52% (by weight) residual oil. In the 25 g/L cellulosecondition, 87.6 pounds of oil (including solids in the oil) wasrecovered. The pressed cake was analyzed for residual oil content andthe cake contained 10-11% (by weight) residual oil. No oil was recoveredfrom the 50 g/L cellulose added condition. The biomass did not feedthrough the press and clogged the press after 5 minutes.

The results from this experiment were consistent with the results fromthe lab-scale bench top press. Although a modest amount of oil wasrecovered from the negative control condition, there was still asignificant amount of residual oil left in the pressed cake. The 25 g/Lcellulose condition performed the best, yielding the most about of oilwith the least amount of residual oil in the pressed cake. The 50 g/Lcellulose condition failed to yield any oil and clogged the press after5 minutes of running These results showed that the addition of apress-aid to the fermentation broth of the harvested microalgal biomassand the co-dried can increase the oil yield when pressed in an oil seedscrew press.

Pilot Scale Two-Step Full Press of Prototheca moriformis

Two-step full press of Prototheca moriformis biomass was undertakenwhereby dried, conditioned Prototheca moriformis biomass was pressed inan expeller press and the spent biomass was then conditioned for asecond time and pressed in an expeller press for a second time.

Prototheca moriformis (UTEX 1435) containing approximately 62% oil (bydry cell weight) was drum dried using the methods described above inExample 3. After drum drying, the moisture content of the biomass wasabout 2.7%. The dried biomass was then heat-conditioned using methodsdescribed in Example 3. The moisture content of the biomass afterheat-conditioning was approximately 1.7-2.1%.

A first pass pressing was performed on the heat-conditioned biomassusing a 3.5″ oil seed screw press (French Oil Mill Company, Piqua, Ohio)with the cage preheated to 194-220° F. Approximately 77.1% of the oil(by weight) was recovered from the microalgal biomass during this firstpass. Analysis of the pressed cake showed that there was about 21% byweight residual oil.

The pressed cake was heat-conditioned a second time so that the pressedcake was about 166-197° F. The moisture content of the heat-conditionedpressed cake was approximately 1.8%. The heat-conditioned cake was thenfed into the press with the cage preheated to 180-235° F. Approximately72.5% of the oil in the cake was recovered in this second pass, based onthe weight of the oil recovered in this second, the pressed cake fromthe second press and the calculated available oil in the pressed cakeafter the first pass through the screw press. By adding the oilrecovered in both passes through the press, the total oil recoveryachieved with both passes was approximately 92.9%.

Monosaccharide Composition of Delipidated Prototheca moriformis Biomass

High oil Prototheca moriformis biomass was then harvested and driedusing a drum dryer. The dried algal biomass was lysed and the oilextracted using an expeller press as described herein. The residual oilin the pressed biomass was then solvent extracted using petroleum ether.Residual petroleum ether was evaporated from the delipidated meal usinga Rotovapor (Buchi Labortechnik AG, Switzerland). Glycosyl(monosaccharide) composition analysis was then performed on thedelipidated meal using combined gas chromatography/mass spectrometry(GC/MS) of the per-O-trimethylsily (TMS) derivatives of themonosaccharide methyl glycosides produced from the sample by acidicmethanolysis. A sample of delipidated meal was subjected to methanolysisin 1M HCl in methanol at 80° C. for approximately 20 hours, followed byre-N-acetylation with pyridine and acetic anhydride in methanol (fordetection of amino sugars). The samples were thenper-O-trimethylsiylated by treatment with Tri-Sil (Pierce) at 80° C. for30 minutes (see methods in Merkle and Poppe (1994) Methods Enzymol. 230:1-15 and York et al., (1985) Methods Enzymol. 118:3-40). GC/MS analysisof the TMS methyl glycosides was performed on an HP 6890 GC interfacedto a 5975b MSD, using a All Tech EC-1 fused silica capillary column (30m×0.25 mm ID). The monosaccharides were identified by their retentiontimes in comparison to standards, and the carbohydrate character ofthese are authenticated by their mass spectra. 20 micrograms per sampleof inositol was added to the sample before derivatization as an internalstandard. The monosaccharide profile of the delipidated Protothecamoriformis (UTEX 1435) biomass is summarized in Table 15 below.

TABLE 15 Monosaccharide (glycosyl) composition analysis of Protothecamoriformis (UTEX 1435) delipidated biomass. Mass Mole % (of total (μg)carbohydrate) Arabinose 0.6 1.2 Xylose n.d. n.d. Galacturonic acid(GalUA) n.d. n.d. Mannose 6.9 11.9 Galactose 14.5 25.2 Glucose 35.5 61.7N Acetyl Galactosamine (GalNAc) n.d. n.d. N Acetyl Glucosamine (GlcNAc)n.d. n.d. Heptose n.d. n.d. 3 Deoxy-2-manno-2 Octulsonic n.d. n.d. acid(KDO) Sum 57 100 n.d. = none detected

Two samples of delipidated Prototheca moriformis (UTEX 1435) biomass wasalso analyzed for dietary fiber content using AOAC Method 991.43. Thedietary fiber content for the two samples was 22.89% and 33.06%.

EXAMPLE 10

Solvent Extraction of Pressed Cake from Microalgal Biomass

To maximize the total oil yield from the microalgal biomass, the pressedcake (as described in the previous Examples) was subjected to solventextraction using a drum batch-type extractor and commercial hexane asthe solvent. The pressed biomass was mixed with the commercial hexane inthe extractor. Extraction of oil was performed in the drum extractor bywashing the pressed cake three times with commercial hexane using asolvent to solids ratio of between 0.7:1 to 2:1. The temperature of theextractor was held at between 122° F. to 131° F., for a residence timeof 1 hour for each wash and a slight vacuum of 1 to 2 inches of water.The drum extractor was rotated continuously during each wash toimproving mixing the extraction efficiency.

The oil-hexane miscella leaving the extractor was filtered through a onemicron filter and then evaporated to a minimum solvent content in abatch evaporation vessel. The solvent was removed by evaporation at 170°F. to 200° F. and a vacuum of 20 to 24 inches of Hg. 0.5% to 2% nitrogenwas sparged to achieve low residual solvent in the crude oil. Thedesolventized crude oil was then packed in 5 gallon containers. The wetspent meal (“marc”) was desolventized in the same drum extractor vesselafter the oil-hexane miscella was pumped out. Desolventization anddrying of the marc was performed in the drum extractor by heating thebiomass to 220° F. to 240° F. using only indirect steam. Thedesolventized meal was packed in 44 gallon fiber drums.

Solvent vapors from the drum extractor and the oil evaporator werecondensed and recovered in the solvent-water tank where the water andsolids were removed. The reclaimed solvent was stored and can be reusedin future solvent extractions.

EXAMPLE 11

Drying and Oil Extraction from Oleaginous Yeast

Oleaginous yeast strain Rhodotorula glutinis (DSMZ-DSM 70398) wascultured according to the methods in Example 1 to produce oleaginousyeast biomass with approximately 50% lipid by DCW. The harvested yeastbroth was dried using three different methods for comparison: (1) traydried in a forced air oven at 75° C. overnight; (2) dried on a drumdryer without concentration; and (3) the yeast broth was concentrated to22% solids and the slurry was then dried on a drum dryer. Material fromeach of the three different drying conditions was heat conditioned andfed through a screw press for oil extraction. The press temperature wasat 150° F. and the conditioned dried yeast biomass was held at about190° F. until it was ready to be fed into the press.

The moisture content of the drum dried yeast broth without concentrationwas 5.4% and the drum dried yeast was then conditioned in an oven at 90°C. for 20 minutes. The moisture content after conditioning was 1.4%. Theconditioned drum dried yeast was then fed into a bench-top Taby screwpress for oil extraction. This material oiled well, with minimalfooting.

The moisture content of the drum dried concentrated yeast broth was 2.1%and the drum dried concentrated yeast was then conditioned in an oven at90° C. for 20 minutes. The moisture content after conditioning was 1.0%.The conditioned drum dried concentrated yeast was then fed into abench-top Taby screw press for oil extraction. This material oiled well,with minimal footing.

EXAMPLE 12

Drying and Oil Extraction from Oleaginous Bacteria

Oleaginous bacteria strain Rhodococcus opacus PD630 (DSMZ-DSM 44193) wascultured according to the methods in Example 1 to produce oleaginousbacteria biomass with approximately 32% lipid by DCW.

The harvested Rhodococcus opacus broth was concentrated usingcentrifugation and then washed with deionized water and resuspended in1.8L of deionized water. 50 grams of purified cellulose(PB20-Pre-co-Floc, EP Minerals, Nevada) was added to the resuspendedbiomass and the total solids was adjusted with deionized water to 20%.The Rhodococcus biomass was then dried on a drum drier and the moisturecontent of the Rhodococcus after drum drying was approximately 3%.

The drum-dried material was then heat conditioned in a oven at 130° C.for 30 minutes with a resulting moisture content of approximately 1.2%.The heat conditioned biomass was then fed through a bench top Taby press(screw press) for oil extraction. The press temperature was at 209° F.and the conditioned dried yeast biomass was held at about 240° F. untilit was ready to be fed into the press. Oil recovery was accompanied byheavy footing.

EXAMPLE 13

Genotyping Microalgal Strains

Microalgae samples from the 23 strains listed in Table 5 above weregenotyped. Genomic DNA was isolated from algal biomass as follows. Cells(approximately 200 mg) were centifuged from liquid cultures 5 minutes at14,000×g. Cells were then resuspended in sterile distilled water,centrifuged 5 minutes at 14,000×g and the supernatant discarded. Asingle glass bead ˜2 mm in diameter was added to the biomass and tubeswere placed at −80° C. for at least 15 minutes. Samples were removed and150 μl of grinding buffer (1% Sarkosyl, 0.25 M Sucrose, 50 mM NaCl, 20mM EDTA, 100 mM Tris-HCl, pH 8.0, RNase A 0.5 ug/ul) was added. Pelletswere resuspended by vortexing briefly, followed by the addition of 40 ulof 5M NaCl. Samples were vortexed briefly, followed by the addition of66 μl of 5% CTAB (Cetyl trimethylammonium bromide) and a final briefvortex. Samples were next incubated at 65° C. for 10 minutes after whichthey were centrifuged at 14,000×g for 10 minutes. The supernatant wastransferred to a fresh tube and extracted once with 300 μl of Phenol:Chloroform:Isoamyl alcohol 12:12:1, followed by centrifugation for 5minutes at 14,000×g. The resulting aqueous phase was transferred to afresh tube containing 0.7 vol of isopropanol (˜190 μl), mixed byinversion and incubated at room temperature for 30 minutes or overnightat 4° C. DNA was recovered via centrifugation at 14,000×g for 10minutes. The resulting pellet was then washed twice with 70% ethanol,followed by a final wash with 100% ethanol. Pellets were air dried for20-30 minutes at room temperature followed by resuspension in 50 μl of10 mM TrisCl, 1 mM EDTA (pH 8.0).

Five μl of total algal DNA, prepared as described above, was diluted1:50 in 10 mM Tris, pH 8.0. PCR reactions, final volume 20 μl, were setup as follows. Ten μl of 2× iProof HF master mix (BIO-RAD) was added to0.4 μl primer SZ02613 (5′-TGTTGAAGAATGAGCCGGCGAC-3′ (SEQ ID NO:24) at 10mM stock concentration). This primer sequence runs from position 567-588in Gen Bank accession no. L43357 and is highly conserved in higherplants and algal plastid genomes. This was followed by the addition of0.4 μl primer SZ02615 (5′-CAGTGAGCTATTACGCACTC-3′ (SEQ ID NO:25) at 10mM stock concentration). This primer sequence is complementary toposition 1112-1093 in Gen Bank accession no. L43357 and is highlyconserved in higher plants and algal plastid genomes. Next, 5 μl ofdiluted total DNA and 3.2 μl dH₂O were added. PCR reactions were run asfollows: 98° C., 45″; 98° C., 8″; 53° C., 12″; 72° C., 20″ for 35 cyclesfollowed by 72° C. for 1 min and holding at 25° C. For purification ofPCR products, 20 μl of 10 mM Tris, pH 8.0, was added to each reaction,followed by extraction with 40 μl of Phenol:Chloroform:isoamyl alcohol12:12:1, vortexing and centrifuging at 14,000×g for 5 minutes. PCRreactions were applied to S-400 columns (GE Healthcare) and centrifugedfor 2 minutes at 3,000×g. Purified PCR products were subsequently TOPOcloned into PCR8/GW/TOPO and positive clones selected for on LB/Specplates. Purified plasmid DNA was sequenced in both directions using M13forward and reverse primers. Sequences from the 23 microalgal strainsare listed as SEQ ID NOs:1-23 in the attached Sequence Listing (seeTable 5 for the correlation). Additionally, several Prototheca strainsof microalgae (see Table 16, below) were also genotyped using themethods and primers described above. 23S rRNA genomic sequences arelisted as SEQ ID NOs:26-34 in the attached Sequence Listing and aredescribed below.

TABLE 16 Prototheca microalgal strains. Species Strain SEQ ID NO.Prototheca kruegani UTEX 329 SEQ ID NO: 26 Prototheca wickerhamii UTEX1440 SEQ ID NO: 27 Prototheca stagnora UTEX 1442 SEQ ID NO: 28Prototheca moriformis UTEX 288 SEQ ID NO: 29 Prototheca moriformis UTEX1439; 1441; SEQ ID NO: 30 1435; 1437 Prototheca wikerhamii UTEX 1533 SEQID NO: 31 Prototheca moriformis UTEX 1434 SEQ ID NO: 32 Protothecazopfii UTEX 1438 SEQ ID NO: 33 Prototheca moriformis UTEX 1436 SEQ IDNO: 34Genotyping Oleaginous Yeast Strains

Genotyping of 48 different strains of oleaginous yeast was performed.Genomic DNA was isolated from each of the 48 different strains ofoleaginous yeast biomass as follows. Cells (approximately 200 mg) werecentrifuged from liquid cultures 5 minutes at 14,000×g. Cells were thenresuspended in sterile distilled water, centrifuged 5 minutes at14,000×g and the supernatant discarded. A single glass bead ˜2 mm indiameter was added to the biomass and tubes were placed at −80° C. forat least 15 minutes. Samples were removed and 150 μl of grinding buffer(1% Sarkosyl, 0.25 M Sucrose, 50 mM NaCl, 20 mM EDTA, 100 mM Tris-HCl,pH 8.0, RNase A 0.5 ug/ul) was added. Pellets were resuspended byvortexing briefly, followed by the addition of 40 ul of 5M NaCl. Sampleswere vortexed briefly, followed by the addition of 66 μl of 5% CTAB(Cetyl trimethylammonium bromide) and a final brief vortex. Samples werenext incubated at 65° C. for 10 minutes after which they werecentrifuged at 14,000×g for 10 minutes. The supernatant was transferredto a fresh tube and extracted once with 300 μl ofPhenol:Chloroform:Isoamyl alcohol 12:12:1, followed by centrifugationfor 5 minutes at 14,000×g. The resulting aqueous phase was transferredto a fresh tube containing 0.7 vol of isopropanol (˜190 μl), mixed byinversion and incubated at room temperature for 30 minutes or overnightat 4° C. DNA was recovered via centrifugation at 14,000×g for 10minutes. The resulting pellet was then washed twice with 70% ethanol,followed by a final wash with 100% ethanol. Pellets were air dried for20-30 minutes at room temperature followed by resuspension in 50 μl of10 mM TrisCl, 1 mM EDTA (pH 8.0).

Five μl of total algal DNA, prepared as described above, was diluted1:50 in 10 mM Tris, pH 8.0. PCR reactions, final volume 20 μl, were setup as follows. Ten μl of 2× iProof HF master mix (BIO-RAD) was added to0.4 μl primer SZ5434 forward primer (5′ GTCCCTGCCCTTTGTACACAC -3′ (SEQID NO: 35) at 10 mM stock concentration) and 0.4 μl primer SZ5435reverse primer (5′-TTGATATGCTTAAGTTCAGCGGG -3′ (SEQ ID NO: 36) at 10 mMstock concentration). The primers were selected based on sequenceconservation between three prime regions of 18S and five prime regionsof fungal 26S rRNA genes. By reference, the forward primer is identicalto nucleotides 1632-1652 of Genbank Ascension #AY550243 and the reverseprimer is identical to nucleotides 464271-464293 of Genbank Ascension#NC_001144. Next, 5 μl of diluted total DNA and 3.2 μl dH₂O were added.PCR reactions were run as follows: 98° C., 45 seconds; 98° C., 8seconds; 58° C., 12 seconds; 72° C., 36 seconds for 35 cycles followedby 72° C. for 1 min and holding at 4° C. For purification of PCRproducts, 20 μl of 10 mM Tris, pH 8.0, was added to each reaction,followed by extraction with 40 μl of Phenol: Chloroform:isoamyl alcohol12:12:1, vortexing and centrifuging at 14,000×g for 5 minutes. PCRreactions were applied to S-400 columns (GE Healthcare) and centrifugedfor 2 minutes at 3,000×g. The resulting purified PCR products werecloned and transformed into E. coli using ZeroBlunt PCR4Blunt-TOPOvector kit (Invitrogen) according to manufacture's instructions.Sequencing reactions were carried out directly on ampicillin resistantcolonies. Purified plasmid DNA was sequenced in both directions usingM13 forward and reverse primers.

A list of the 48 strains of oleaginous yeast that were genotyped issummarized below in Table 17 along with the corresponding SEQ ID NOs.

TABLE 17 Oleaginous yeast strains. Strain Name Strain Number SEQ ID NORhodotorula glutinis DSMZ-DSM SEQ ID NO: 37 70398 Lipomyces tetrasporusCBS 5911 SEQ ID NO: 37 Rhodotorula glutinis var. glutinis CBS 3044 SEQID NO: 38 Lipomyces tetrasporus CBS 8664 SEQ ID NO: 38 Lipomycestetrasporus CBS 1808 SEQ ID NO: 39 Lipomyces tetrasporus CBS 1810 SEQ IDNO: 39 Lipomyces starkeyi CBS 1809 SEQ ID NO: 40 Trichosporonmontevideense CBS 8261 SEQ ID NO: 40 Yarrowia lipolytica CBS 6331 SEQ IDNO: 41 Cryptococcus curvatus CBS 5324 SEQ ID NO: 42 Rhodotorulamucilaginosa var. CBS 316 SEQ ID NO: 42 mucilaginosa Cryptococcuscurvatus CBS 570 SEQ ID NO: 42 Cryptococcus curvatus CBS 2176 SEQ ID NO:42 Cryptococcus curvatus CBS 2744 SEQ ID NO: 42 Cryptococcus curvatusCBS 2754 SEQ ID NO: 42 Cryptococcus curvatus CBS 2829 SEQ ID NO: 42Cryptococcus curvatus CBS 5163 SEQ ID NO: 42 Cryptococcus curvatus CBS5358 SEQ ID NO: 42 Trichosporon sp. CBS 7617 SEQ ID NO: 43Sporobolomyces alborubescens CBS 482 SEQ ID NO: 44 Rhodotorula glutinisvar. glutinis CBS 324 SEQ ID NO: 45 Rhodotorula glutinis var. glutinisCBS 4476 SEQ ID NO: 46 Trichosporon behrend CBS 5581 SEQ ID NO: 47Geotrichum histeridarum CBS 9892 SEQ ID NO: 48 Rhodotorula aurantiacaCBS 8411 SEQ ID NO: 49 Cryptococcus curvatus CBS 8126 SEQ ID NO: 49Trichosporon domesticum CBS 8111 SEQ ID NO: 50 Rhodotorula toruloidesCBS 8761 SEQ ID NO: 51 Rhodotorula terpendoidalis CBS 8445 SEQ ID NO: 52Yarrowia lipolytica CBS 10144 SEQ ID NO: 53 Rhodotorula glutinis var.glutinis CBS 5805 SEQ ID NO: 54 Yarrowia lipolytica CBS 10143 SEQ ID NO:55 Lipomyces tetrasporus CBS 5607 SEQ ID NO: 56 Yarrowia lipolytica CBS5589 SEQ ID NO: 57 Lipomyces tetrasporus CBS 8724 SEQ ID NO: 58Rhodosporidium sphaerocarpum CBS 2371 SEQ ID NO: 59 Trichosporonbrassicae CBS 6382 SEQ ID NO: 60 Cryptococcus curvatus CBS 2755 SEQ IDNO: 61 Lipomyces tetrasporus CBS 7656 SEQ ID NO: 61 Lipomyces starkeyiCBS 7786 SEQ ID NO: 62 Yarrowia lipolytica CBS 6012 SEQ ID NO: 63Trichosporon loubieri var. loubieri CBS 8265 SEQ ID NO: 64 Geotrichumvulgare CBS 10073 SEQ ID NO: 65 Rhodosporidium toruloides CBS 14 SEQ IDNO: 66 Rhodotorula glutinis var. glutinis CBS 6020 SEQ ID NO: 67Lipomyces orientalis CBS 10300 SEQ ID NO: 67 Rhodotorula aurantiaca CBS317 SEQ ID NO: 68 Torulaspora delbrueckii CBS 2924 SEQ ID NO: 69

All references cited herein, including patents, patent applications, andscientific journal publications, are hereby incorporated by reference intheir entireties, whether previously specifically incorporated or not.In particular, PCT Application No. PCT/US2010/031088, filed Apr. 14,2010, entitled “Novel Microalgal Food Compositions” is incorporatedherein by reference. The publications mentioned herein are cited for thepurpose of describing and disclosing reagents, methodologies andconcepts that may be used in connection with the present invention.Nothing herein is to be construed as an admission that these referencesare prior art in relation to the inventions described herein.

Although this invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications. The following claims are intended to cover anyvariations, uses or adaptations of the invention following, in general,the principles of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth.

What is claimed is:
 1. A method for extracting triglyceride oil frommicroalgal biomass, the method consisting of: a. drying the microalgalbiomass to produce dried microalgal biomass; b. conditioning the driedmicroalgal biomass by heating the dried microalgal biomass to atemperature of from 70° C. to 150° C. to produce conditioned feedstockhaving a moisture content of from 0.5% to 2.5% by weight of themicroalgal biomass wherein prior to said conditioning a bulking agent isadded to the dried microalgal biomass; and c. subjecting the conditionedfeedstock having a moisture content of from 0.5% to 2.5% to pressuresufficient to separate at least 25% of the total triglyceride oil fromthe conditioned feedstock, leaving spent microalgal biomass of reducedtriglyceride oil content relative to the conditioned feedstock, whereinsaid method does not use organic solvent for the extraction.
 2. Themethod of claim 1, wherein the triglyceride oil is extracted by anexpeller press.
 3. The method of claim 1, wherein more than 50% of thetriglyceride oil is separated from the conditioned feedstock.
 4. Themethod of claim 2, wherein the expeller press is a continuously rotatingworm shaft within a cage having a feeder at one end and a choke at anend opposite thereof, and having openings within the cage, wherein thecage has an internal length that is between at least ten times to atleast 20 times its internal diameter and comprises a plurality ofelongated bars with at least some of the elongated bars separated by oneor more spacers, the bars resting on a frame, wherein the one or morespacers between the bars form the openings, and triglyceride oil isreleased through the openings to a collecting vessel fluidly coupledwith the cage, wherein the spacers between the elongated bars are ofdifferent thicknesses thereby allowing variation of the space betweeneach elongated bar, wherein either the spacers or the gaps between thebars are from 0.005 to 0.030 inches thick, wherein the biomass entersthe cage through the feeder, and rotation of the worm shaft advances thebiomass along the cage and applies pressure to the biomass disposedbetween the cage and the choke, the pressure lysing cells of the biomassand releasing triglyceride oil through the openings of the cage, andspent biomass of reduced triglyceride oil content is extruded from thechoke end of the cage.
 5. The method of claim 4, wherein the pressureincreases by a factor of between 10 and 20 from the feeder end to thechoke end of the cage and does not increase by more than 100% of thepressure at the feeder end of the cage per linear foot of the cagebetween the feeder and choke ends of the cage.
 6. The method of claim 1,wherein the moisture content of the conditioned feedstock is between0.7% and 1.2% by weight.
 7. The method of claim 1, wherein the biomassis conditioned with heat for a period of time between 10 and 60 minutes.8. The method of claim 7, wherein a bulking agent is switchgrass,soybean hulls, dried rosemary, corn stover, cellulose, sugar canebagasse, or spent microbial biomass that comprises between 40% and 90%polysaccharide and less than 10% triglyceride oil, and is added to themicrobial biomass prior to conditioning the dried microbial biomass. 9.The method of claim 1, wherein the microalgal biomass that wascultivated through a process selected from the group consisting of aheterotrophic process, a photoautotrophic process, and a mixotrophicprocess.
 10. The method of claim 1, wherein the microalgal biomass has afatty acid profile of at least 4% C8-C14.
 11. The method of claim 1,wherein the extracted triglyceride oil has one or more propertiesselected from: less than 0.01 milligram of chlorophyll per kilogram oftriglyceride oil; or one or more of the following: no more than 8 ppmchloride, no more than 2 ppm phosphorus, no more than 26 ppm potassium,no more than 12 ppm sodium, and no more than 5 ppm sulfur.
 12. Themethod of claim 1, wherein the spent biomass comprises less than 40%triglyceride oil by weight.
 13. The method of claim 12, wherein thespent biomass comprises less than 20% triglyceride oil by weight. 14.The method of claim 13, wherein the spent biomass comprises less than10% triglyceride oil by weight.
 15. The method of claim 14, wherein thespent biomass comprises less than 5% triglyceride oil by weight.
 16. Themethod of claim 1, wherein the pressure is applied mechanically.
 17. Themethod of claim 1, wherein the pressure that is applied mechanically isapplied by an expeller press.
 18. A method for extracting triglycerideoil from microalgal biomass, the method consisting of: a. drying themicroalgal biomass to produce dried microalgal biomass; b. conditioningthe dried microalgal biomass by heating to a temperature of from 70° C.to 150° C. to produce conditioned feedstock having a moisture content offrom 0.5% to 2.5% by weight of the microalgal biomass wherein prior tosaid conditioning a bulking agent is added to the dried microalgalbiomass; and c. subjecting the conditioned feedstock having a moisturecontent of from 0.5% to 2.5% to pressure sufficient to separate at least5% of the total triglyceride oil from the conditioned feedstock andleaving spent biomass of reduced triglyceride oil content relative tothe conditioned feedstock, wherein said method does not use organicsolvent for the extraction.
 19. The method of claim 18, wherein themicroalgal biomass is derived from microalgae of the genus Chlorella orthe genus Prototheca.
 20. The method of claim 19, wherein the moisturecontent is from 0.5% to 2.0% by weight.
 21. The method of claim 20,wherein the moisture content is from 0.7% to 1.2% by weight.
 22. Themethod of claim 18, wherein the pressure is applied mechanically. 23.The method of claim 22, wherein the pressure that is appliedmechanically is applied by a expeller press.